Acetylation at lysine 71 inactivates superoxide dismutase 1 and sensitizes cancer cells to genotoxic agents

Abstract

Cancer cells are characterized by a high dependency on antioxidant enzymes to cope with the elevated rates of reactive oxygen species (ROS). Impairing antioxidant capacity in cancer cells disturbs the ROS homeostasis and exposes cancer cells to massive oxidative stress. In this study, we have discovered that superoxide dismutase 1 (SOD1), a major player in maintaining the cellular redox status, was acetylated at lysine 71. This acetylation, which was primarily deacetylated by Sirtuin 1 (SIRT1), suppressed the enzymatic activity of SOD1 via disrupting its association with copper chaperone for SOD1 (CCS). More importantly, genotoxic agents, such as camptothecin (CPT), induced SOD1 acetylation by disrupting its binding with SIRT1. CPT-induced SOD1 acetylation was stimulated by its provoked ROS, suggesting a positive feedback loop, in which ROS per se impairs the antioxidative defence of cancer cells and reinforces oxidative stress stimulated by anticancer agents. The intrinsic abundance of SOD1 acetylation varied among cancer cells, and high level of SOD1 acetylation was correlated with elevated sensitivity to CPT. Together, our findings gained mechanistic insights into how cytotoxic agents fine tune the intracellular ROS homeostasis to strengthen their anticancer effects, and suggested SOD1 acetylation as a candidate biomarker for predicting response to CPT-based chemotherapy.

Keywords: SIRT1; acetylation; camptothecin; oxidative stress; superoxide dismutase.

Figure 1. SOD1 is acetylated on K71 residue

A. Acetylation of exogenous SOD1. Ectopically expressed SOD1 was immunopreciptated and acetylation of SOD1 was examined using a pan-acetyl-lysine antibody. Cells were pretreated with TSA (500 nM) or/and NAM (10 mM) for 12 hr prior to immunoprecipitation; B., C. Impact of lysine mutant on SOD1 acetylation. B., wildtype (WT) SOD1 or indicated lysine mutants was transfected into cells and acetylation of each mutant was examined as described in A.. EV, empty vector; C., Acetylation of ectopically expressed SOD1WT, SOD1K71R, SOD1K71Q with or without TSA (500 nM) and NAM (10 mM) treatment was analyzed; D. Specificity of anti-acetyl-K71 antibody. Flag-tagged SOD1 WT or K71R mutant was transfected into HCT116 cells and acetylation of each purified protein was measured by immunoblotting using an anti-Ac SOD1 (K71) antibody; E. Endogenous SOD1 is acetylated at K71. Cells treated with NAM and TSA was analyzed by anti-Ac SOD1 (K71) antibody.

Figure 2. SOD1 acetylation inactivates SOD1

A. SOD1 activity is decreased by NAM/TSA treatment. Cells were treated by TSA (500 nM) and NAM (10 mM) for 12 hr and SOD1 activity was determined using an in-gel SOD1 activity assay; B. Mutations of the acetylation lysine residues affect SOD1 activity. Flag-tagged SOD1 WT, K71R or K71Q mutant was transfected into HCT116 cells and SOD1 activity was measured as in A.. endo, endogenous SOD1, exo, Flag-tagged SOD1; C. Sufficient supply of copper rescues the activity of K711Q mutant; D. The interaction between SOD1 and CCS is decreased by NAM/TSA treatment. HCT-116 cells transfected with Flag-tagged SOD1 were treated with NAM/TSA for 12 hr. The presence of CCS in the immunoprecipitated protein complexes was assessed by immunoblotting; E. Acetylation mimetic mutation at K71 decreased the interaction between SOD1 and CCS; F. The level of SOD1 homodimers is reduced under NAM/TSA treatment. HCT116 cells were treated with NAM/TSA for 12 hr. Cells lysis in the presence or absence of DMP (20 mM) were assessed by immunoblotting using SOD1 antibody; G. Acetylation mimetic mutation at K71 decreased the level of SOD1 homodimers. HCT116 cells stably knock-down SOD1 (shSOD1) were transfected with Flag-tagged SOD1 WT, SOD1K71R or SOD1K71Q. SOD1 homodimers were detected as described in C..

Figure 3. SIRT1 deacetylates SOD1

A. SIRT1 knockdown increased SOD1 acetylation. Cells were transfected with siRNA against SIRT1 to 7 and SOD1 acetylation was detected by anti-Ac SOD1 (K71) antibody; The Ac-SOD1 band was indicated by the solid triangle. B. SIRT1 deacetylated SOD1 acetylation on K71. HCT116 ShSIRT1 cells were transfected with flag-tagged SOD1 wildtype (WT) or K71R mutant. SOD1 acetylation was detected by using a pan-acetyl-lysine antibody following anti-Flag immunoprecipitation; C. Catalytic activity of SIRT1 is required for the deacetylation of SOD1. HA-tagged SOD1 was co-transfected with Flag-tagged SIRT1 wildtype (WT) or catalytically inactive mutant H363Y into SIRT1-depleted HCT-116 cells. Acetylation was detected as in B., D. Catalytic activity of SIRT1 is required for the regulation of SOD1 activity. Endogenous SOD1 activity was measured in shSIRT1 cells reconstituted with SIRT1 WT or H363Y mutant; E., F. SOD1 interacts with SIRT1. E, HA-tagged SOD1 was co-transfected with Flag-tagged SIRT1 into HCT-116 cells. Co-immunoprecipitation was performed using anti-Flag agarose. The presence of SOD1 in the immunoprecipitate was assessed by immunoblotting. F, interaction of endogenous SOD1 and SIRT1. Endogenous SOD1 was immunoprecipitated with a rabbit polyclonal antibody against SOD1. Rabbit Immunoglobulin G (IgG) was used as a negative control. The presence of SIRT1 in the immunoprecipitate was assessed by immunoblotting with an antibody against SIRT1.

Figure 4. Genotoxic agents promote SOD1 acetylation and inactivate SOD1

A., Genotoxic agent induces SOD1 acetylation. HCT-116 were treated with camptothecin (CPT, 100 nM), etoposide(VP-16, 10 μM), bleomycin (BLM, 10 μM) or cisplatin (CDDP, 20 μM) for 12 hr and SOD1 acetylation was examined using an anti-Ac SOD1 (K71) antibody; B. CPT-induced SOD1 acetylation is mediated by ROS. Cells were pretreated with or without NAC or APO for 2 hr before exposure to CPT (100 nM, 12 hr); C., D. The impact of CPT treatment on SOD1 acetylation and enzymatic activity. Time-dependent C. and dose-dependent D. induction of SOD1 acetylation and corresponding alteration in SOD1 activity was measured using immunoblotting or an in-gel enzymatic assay; E. The impact of CPT treatment on the interaction between SOD1 and CCS. Flag-tagged SOD1 WT, K71R, K71Q was transfected into HCT116 cells treated with or without 100 nM CPT for 12 hr. The presence of CCS in the immunoprecipitated protein complexes was assessed by immunoblotting. F. The impact of CPT on the formation of SOD1 homodimers. Flag-tagged SOD1 WT, K71R, K71Q was transfected into HCT116 cells treated with or without 100 nM CPT for 12 hr; G. The impact of CPT on the interaction between SOD1 and SIRT1. HA-tagged SOD1 were co-transfected with Flag-tagged SIRT1 into HCT-116 cells; H. The impact of SOD1 acetylation on cytosolic superoxide levels under CPT stimulation. HCT116 cells stably knock-down SOD1 (shSOD1) were transfected with Flag-tagged SOD1 (WT), SOD1K71R or SOD1K71Q, and cells were treated with CPT. The cytosolic superoxide level was measured by DHE staining and FACS analysis. Data represent mean ± SEM (n = 3), **P < 0.01. I. SOD1 Acetylation promotes cells sensitive to CPT treatment. The sensitivity of cells HCT116 shSOD1 cells expressing the wildtype, K71R or K71Q SOD1 were treated with CPT (1 nM) for 14 days and measured by a cologeneic assay.

Figure 5. SOD1 acetylation is associated with the response to CPT treatment

A. SOD1 acetylation and cell sensitivity to CPT-11 in colon cancer cell lines. B., C. Intratumoral SOD1 acetylation level and response to CPT-11 treatment of patient-derived xenograft models. B. Growth curves of tumors from indicated PDX models treated with vehicle control or 10 mg/kg CPT-11 daily for 21 consecutive days. Data represent mean tumor volume ± SEM (n = 6), ***P < 0.001; *P < 0.05; n.s., not significant. C. Correlation between SOD1 K71 acetylation and sensitivity to CPT-11. T/C%, relative tumor volume versus vehicle control on day 21.

Figure 6. A schematic model showing the positive feedback of ROS induction resulted from SOD1 acetylation