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. 2014 Jan;124(1):117-28.
doi: 10.1172/JCI71714.

Targeting SOD1 reduces experimental non–small-cell lung cancer

Andrea Glasauer, Laura A Sena, Lauren P Diebold, Andrew P Mazar, Navdeep S Chandel

Targeting SOD1 reduces experimental non–small-cell lung cancer

Andrea Glasauer et al. J Clin Invest. 2014 Jan.

Abstract

Approximately 85% of lung cancers are non–small-cell lung cancers (NSCLCs), which are often diagnosed at an advanced stage and associated with poor prognosis. Currently, there are very few therapies available for NSCLCs due to the recalcitrant nature of this cancer. Mutations that activate the small GTPase KRAS are found in 20% to 30% of NSCLCs. Here, we report that inhibition of superoxide dismutase 1 (SOD1) by the small molecule ATN-224 induced cell death in various NSCLC cells, including those harboring KRAS mutations. ATN-224–dependent SOD1 inhibition increased superoxide, which diminished enzyme activity of the antioxidant glutathione peroxidase, leading to an increase in intracellular hydrogen peroxide (H(2)O(2)) levels. We found that ATN-224–induced cell death was mediated through H(2)O(2)-dependent activation of P38 MAPK and that P38 activation led to a decrease in the antiapoptotic factor MCL1, which is often upregulated in NSCLC. Treatment with both ATN-224 and ABT-263, an inhibitor of the apoptosis regulators BCL2/BCLXL, augmented cell death. Furthermore, we demonstrate that ATN-224 reduced tumor burden in a mouse model of NSCLC. Our results indicate that antioxidant inhibition by ATN-224 has potential clinical applications as a single agent, or in combination with other drugs, for the treatment of patients with various forms of NSCLC, including KRAS-driven cancers.

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Figures

Figure 1
Figure 1. SOD1 inhibition causes cell death and anchorage-independent growth impairment in human NSCLC cells.
(A) SOD1 activity assay of A549 KRASonc human NSCLC cells treated for 24 hours with 10 μM ATN-224 (n = 3). (B) A549 cells expressing an empty adenoviral control vector or adenoviral SOD1 (Ad SOD1) were treated with increasing doses of ATN-224 (1.25, 2.5, 5, 10, and 20 μM), and cell death (96 hours) was determined (n = 3). Immunoblot for SOD1 of empty or Ad SOD1–expressing cells. Tubulin (Tub) was used as a loading control. (C and D) A549 cells expressing a pLKO.1 lentiviral scrambled (Scr) control vector or two independent SOD1 shRNA vectors (clones 8 and 44) were (C) immunoblotted for SOD1 and (D) analyzed for cell death 96 hours after infection (n = 3). (E and F) Analysis of soft agar colonies of A549 cells (E) expressing pLKO.1 control or SOD1 shRNA vectors (clones 8 and 44) and (F) treated for 3 weeks with 10 μM ATN-224. Images show colony formation in 96-well plates (n = 8, n = 13). (G and H) NBECs (G) expressing pLKO.1 control or SOD1 shRNA vectors (clones 8 and 44) were immunoblotted for SOD1 or (H) treated with 10 μM ATN-224 and analyzed for cell death (96 hours) (n = 3). Data are represented as the mean ± SEM. *P < 0.05. **P < 0.01. See also Supplemental Figure 1.
Figure 2
Figure 2. SOD1 inhibition induces cell death by diminishing antioxidant protein activities, leading to an increase in H2O2.
(A) A549 cells were treated with 10 μM ATN-224 alone or with 100 μM MnTBAP or 20 μM ebselen. ROS levels were determined by DCFH staining (10 μM, 24 hours) (n = 3). (B and C) A549 cells were treated with 10 μM ATN-224, and H2O2 levels were determined by (B) cyto-roGFP or (C) roGFP2-ORP1 oxidation (both 24 hours) (n = 3). (D and E) A549 cells were treated with 10 μM ATN-224 alone or with 100 μM MnTBAP, and (D) cell death (96 hours) and (E) anchorage-independent growth (3 weeks) were determined (n = 3, n = 7). (F) ATN-224–treated A549 cell lysates were taken (48 hours) and immunoblotted for SOD1. (G) A549 cells were treated with 10 μM ATN-224, and GPX activity was determined (48 hours) (n = 4). (H and I) A549 cells were treated with 10 μM ATN-224 alone or with (H) 20 μM ebselen or (I) 5 mM NAC, and cell death was determined (96 hours) (n = 4, n = 3). (J) A549 cells expressing empty adenoviral vector (Ad empty) or adenoviral GPX1 (Ad GPX1) were treated with 10 μM ATN-224, and cell death (96 hours) was determined (n = 7). Immunoblot for GPX1. (K) A549 cells were treated with 10 μM ATN-224 alone or with 100 μM MnTBAP or 20 μM ebselen. Cell lysates were immunoblotted for PRX-SO3. PRX1 was used as a loading control. Data are represented as the mean ± SEM. *P < 0.05. **P < 0.01. See also Supplemental Figure 2.
Figure 3
Figure 3. ATN-224–induced H2O2 downregulates MCL1 protein expression, leading to programmed cell death.
(A) A549 cells were treated with 10 μM ATN-224 alone or with 30 μM caspase inhibitor zVAD, and cell death (96 hours) was determined (n = 3). (B) A549 cells were treated with 10 μM ATN-224. Cell lysates (72 hours) were immunoblotted for CC3. (C–E) Immortalized wild-type or (C) Casp9–/– MEFs, (D) Bax–/–Bak–/– MEFs, and (E) Bim–/–Puma–/– MEFs were treated with 5 μM ATN-224, and cell death (48 hours) was determined (all n = 3). (F) A549 cells expressing a pBABE control or BCLXL vector were treated with 10 μM ATN-224, and cell death (96 hours) was determined (n = 4). Immunoblot for BCLXL of control or BCLXL vector–expressing cells. (G) A549 cells were treated with 10 μM ATN-224, and lysates (48 hours) were immunoblotted for BCLXL. (H) A549 cells were treated with 10 μM ATN-224 alone or with 100 μM MnTBAP or 20 μM ebselen. Lysates (48 hours) were immunoblotted for MCL1. (I) A549 cells expressing a pLNCX control or MCL1 vector were treated with 10 μM ATN-224, and cell death (96 hours) was determined (n = 3). Immunoblot for MCL1 of control or MCL1 vector–expressing cells. Data are represented as the mean ± SEM. *P < 0.05. **P < 0.01. See also Supplemental Figure 3.
Figure 4
Figure 4. ATN-224–mediated increase in H2O2 activates P38 MAPK activity, leading to MCL1 downregulation and cell death.
(A) A549 cells were treated with 10 μM ATN-224 alone or with 100 μM MnTBAP or 20 μM ebselen. Lysates (48 hours) were immunoblotted for phospho(Thr180/Tyr182)-P38 MAPK. Total P38 MAPK was used as a loading control. (B) A549 cells were treated with 10 μM ATN-224 alone or with 10 μM of the P38 inhibitor SB203580, and cell death (96 hours) was determined (n = 9). (C and D) A549 cells were treated with 10 μM ATN-224 alone or with either (C) 10 μM of the JNK inhibitor SP600125 or (D) 2.5 μM of the ERK1/2 inhibitor FR180204. Cell death (96 hours) was determined (n = 4, n = 3). Vehicle and ATN-224 cell lysates (48 hours) were immunoblotted for (C) phospho(Thr183/Tyr185) JNK or (D) phospho(Thr202/Tyr204) ERK1/2. Total JNK and total ERK1/2 were used as loading controls. (E and F) A549 cells were treated with 10 μM ATN-224 alone or with 10 μM of the P38 MAPK inhibitor SB203580. Lysates (48 hours) were immunoblotted for (E) MCL1 or (F) the P38 MAPK target phospho(Thr222)-MAPKAPK-2. Shift of total MAPKAPK-2 (F) to 49 kDa indicates phosphorylation. Data are represented as the mean ± SEM. *P < 0.05. **P < 0.01.
Figure 5
Figure 5. ATN-224–mediated SOD1 inhibition induces cell death in KrasG12D Tp53fl/fl–driven mouse lung cancer cells.
(A) KP cells were treated with increasing doses of ATN-224 (5, 10, 15, and 20 μM), and cell death (96 hours) by trypan blue staining was determined (n = 4). Statistical significance was determined by comparing 0 μM ATN-224 with increasing concentrations. (B) KP cells were treated with 15 μM ATN-224 alone or with 100 μM MnTBAP or 20 μM ebselen, and cell death (96 hours) was determined (n = 3). (C) Analysis of soft agar colonies of KP cells treated with 10 μM ATN-224 alone or with 100 μM MnTBAP (3 weeks) (n = 5). (D) KP cells were treated with 15 μM ATN-224 alone or with 100 μM MnTBAP or 20 μM ebselen. Lysates (48 hours) were immunoblotted for phospho-P38 MAPK (pP38) and MCL1. Total P38 MAPK was used as a loading control. (E) KP cells were treated with 15 μM ATN-224 alone or with 10 μM of the P38 MAPK inhibitor SB203580. Cell death (96 hours) was determined (n = 4). Data are represented as the mean ± SEM. *P < 0.05. **P < 0.01. See also Supplemental Figure 4.
Figure 6
Figure 6. ATN-224 as a single agent reduces tumor burden in a preclinical KrasG12D Tp53fl/fl–driven mouse model of lung cancer.
(A) Schematic of experimental design. KP mice were intubated with 107 PFUs of adenoviral Cre and treated 8 wpi with PBS or 4 mg/kg ATN-224 every 2 days for 4 weeks. (B) KP mice were injected i.p. with PBS or 4 mg/kg ATN-224 for 4 weeks, and ATN-224 levels in blood plasma were determined (n = 3). (C–E) Lungs isolated from PBS and ATN-224–treated KP mice were analyzed for (C) lung weight normalized to body weight, (D) number of lesions per whole lung section, and (E) tumor burden per whole lung section (n = 16 PBS, n = 17 ATN-224). Tumor burden was calculated by averaging the tumor area from H&E-stained whole lung sections shown in H (left panel). (F and G) Lungs isolated from PBS and ATN-224–treated KP mice were analyzed for (F) KI67-positive cells per lung section and (G) CC3-positive cells per lung section (n = 5 PBS, n = 5 ATN-224). (H) Representative images of H&E- (left panels), KI67- (middle panels), and CC3-stained (right panels) KP lung sections from PBS and ATN-224–treated mice. H&E-stained images of whole lung sections represent tumor burden. Scale bars: 1 mm (left panels). KI67 (proliferation) and CC3 (cell death) images of tumors; positively stained cells appear in brown and nuclei in blue. Scale bars: 50 μM (middle and right panels). Data are represented as the mean ± SEM. *P < 0.05. **P < 0.01. See also Supplemental Figure 5.
Figure 7
Figure 7. ATN-224 synergizes with proapoptotic and pro-oxidant drugs to induce augmented cell death in oncogenic KRAS– and KRAS/TP53-driven NSCLC cells.
(A–C) KP cells (A), A549 cells (B), and H460 cells (C) were treated with increasing doses of ATN-224 alone or with the BCL2 inhibitor ABT-263, and cell death (96 hours) was determined (n = 4, n = 3, n = 4). (D–F) KP cells (D), A549 cells (E), and H460 cells (F) were treated with increasing doses of ATN-224 alone or with the glutathione scavenger BSO, and cell death (96 hours) was determined (n = 4, n = 4, n = 4). (G and H) Immortalized wild-type or Bax–/–Bak–/– MEFs were treated with 5 μM ATN-224 alone or with (G) ABT-263 (300 nM) or (H) BSO (10 μM). Cell death (48 hours) was determined (both n = 3). Data are represented as the mean ± SEM. *P < 0.05. **P < 0.01.
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