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

Abstract

SIRT2 is a protein deacetylase with tumor suppressor activity in breast and liver tumors where it is mutated; however, the critical substrates mediating its antitumor activity are not fully defined. Here we demonstrate that SIRT2 binds, deacetylates, and inhibits the peroxidase activity of the antioxidant protein peroxiredoxin (Prdx-1) in breast cancer cells. Ectopic overexpression of SIRT2, but not its catalytically dead mutant, increased intracellular levels of reactive oxygen species (ROS) induced by hydrogen peroxide, which led to increased levels of an overoxidized and multimeric form of Prdx-1 with activity as a molecular chaperone. Elevated levels of SIRT2 sensitized breast cancer cells to intracellular DNA damage and cell death induced by oxidative stress, as associated with increased levels of nuclear FOXO3A and the proapoptotic BIM protein. In addition, elevated levels of SIRT2 sensitized breast cancer cells to arsenic trioxide, an approved therapeutic agent, along with other intracellular ROS-inducing agents. Conversely, antisense RNA-mediated attenuation of SIRT2 reversed ROS-induced toxicity as demonstrated in a zebrafish embryo model system. Collectively, our findings suggest that the tumor suppressor activity of SIRT2 requires its ability to restrict the antioxidant activity of Prdx-1, thereby sensitizing breast cancer cells to ROS-induced DNA damage and cell cytotoxicity. Cancer Res; 76(18); 5467-78. ©2016 AACR.

Figure 1. Knockdown (KD) of SIRT2 by shRNA induces acetylation of proteins including Peroxiredoxin-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 α-tubulin, and α-tubulin. The expression levels of β-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 knockdown cell lines and 2-D differential in-gel electrophoresis (DIGE) was performed. A representative 2D-gel image is presented. Protein spots exhibiting more than a two-fold difference in mobility in SIRT2-KD samples compared to vector control were identified by mass spectrometry. The arrows indicate the location of Peroxiredoxin 1 (Prdx-1), Malate Dehydrogenase (MDH) and Phosphoglycerate Kinase 1 (PGK1).

Figure 2. SIRT2 interacts with peroxiredoxin 1

A. Cells lysates from HEK293 cells expressing FLAG tagged peroxiredoxin-1 (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 (L.C.) 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 lines indicate a repositioned gel lane. D. Cell lysates from MCF7 cells treated with 1mM of nicotinamide (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 β-Actin antibodies. Values underneath the blots indicate densitometry analysis. E. MDA-MB-231 cells transfected with vector, ectopic overexpression of SIRT2 or knockdown 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.

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 β-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 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 X/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 μg) 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 hydrogen peroxide for 2 hours at 37°C. The media was aspirated and cells were washed with 1X PBS. DCF-DA (in phenol red free media) was added to the cells at a final concentration of 10 μM and incubated for 30 min at 37°C. The excess dye was removed by washing with 1X 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 α-tubulin served as loading control.

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 μM of hydrogen peroxide for 0–4 hours. At the end of treatment, the cells were lysed with native lysis buffer and separated on NuPAGE^® 3–8% tris-acetate native gels to detect oligomers and multimers of peroxiredoxin-1 (A, left panel) and oxidized peroxiredoxin 1 (A, right panel). Cell lysates were also probed with anti-β-actin to confirm equal loading. B. MDA-MB-231 vector or SIRT2 O/E cells were treated with 500 μM of hydrogen peroxide for 30 minutes. Then, cell lysates (500 μg) were collected and peroxiredoxin-1 was immunoprecipitated by anti-Peroxiredoxin I (LF-MA0214, AbFrontier) and Dynabeads^® M-280 sheep anti-mouse IgG (Invitrogen). The immunoprecipitated peroxiredoxin-1 and Dynabeads were used for the chaperone activity assay. Each reaction contained 2 μM malate dehydrogenase (MDH) and the immunoprecipitated peroxiredoxin-1 with beads in 200 μL of 50 mM HEPES-KOH buffer (pH 7.5). The absorbance at 320 nm was monitored utilizing a BioTek SynergyMx plate reader at 43 °C for 2 hours. C. MCF7 vector and SIRT2 O/E cells were treated with the indicated concentrations of hydrogen peroxide for 30 minutes. At the end of treatment, the cells were lysed with native lysis buffer and separated on NuPAGE^® 3–8% tris-acetate native gels to detect oligomers and multimers of peroxiredoxin-1 (C, left panel) and oxidized peroxiredoxin-1 (C, right panel). Cell lysates were also probed with anti- β-actin to confirm equal loading. D. MCF7 vector and SIRT2 O/E cells were treated with the indicated concentrations of hydrogen peroxide for 30 minutes. Cell lysates were collected as in (B) and absorbance at 320 nm was monitored utilizing a BioTek SynergyMx plate reader at 43 °C for 2 hours, as above.

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 μM of H₂O₂ for 4 hours. After treatment, the cells were fixed, permeabilized, and blocked with 3% BSA. The expression of γH2AX 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 γH2AX and β-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. * indicates values significantly greater in cells treated with hydrogen peroxide than untreated control cells (p< 0.05).

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 63X/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 hydrogen peroxide (H₂O₂) for 4 hours. * indicate 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 (top panel), or with 500 μM of H₂O₂ for indicated times (bottom panel). The percentages of propidium iodide (PI)-positive, non-viable 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 arsenic trioxide for 48 hours. At the end of treatment, the percentages of PI-positive, non-viable 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, non-viable cells, in each condition was determined by flow cytometry.

Figure 7. Knockdown of SIRT2 in the zebrafish embryos decreased H₂O₂-induced ROS levels and abrogated ROS-mediated cardiac edema and abnormal body curvature

A. The splice-blocking MO targeting against exon 6 (coding for the small domain of SIRT2) of zebrafish SIRT2 was injected into single-cell stage zebrafish embryos, and expression of SIRT2 mRNA was assessed from the embryos, after 48 hours, by qPCR. B. Control and MO treated embryos were exposed to 3 mM of hydrogen peroxide at 48-hpf with or without N-acetyl cysteine (NAC). ROS levels in the embryos were monitored at 30 min using DCF-DA. C. The morphological changes at day 5 (after hydrogen peroxide treatment, 168-hpf) were imaged. D. Schematic model for the activity of increased SIRT2 in breast cancer cells. Induction of SIRT2 decreases the antioxidant activity of Prdx-1, leading to oxidation of Prdx-1 and increased reactive oxygen species (ROS). This induces the transient induction of Prdx-1 multimers and increased Prdx-1 chaperone activity. The ROS that are generated induce the translocation of FOXO3A into the nucleus where it can activate the transcription of pro-apoptotic BIM. The ROS can also directly induce DNA damage, leading to increased cell death.