Superoxide dismutase 1 (SOD1) is a target for a small molecule identified in a screen for inhibitors of the growth of lung adenocarcinoma cell lines

Abstract

We previously described four small molecules that reduced the growth of lung adenocarcinoma cell lines with either epidermal growth factor receptor (EGFR) or KRAS mutations in a high-throughout chemical screen. By combining affinity proteomics and gene expression analysis, we now propose superoxide dismutase 1 (SOD1) as the most likely target of one of these small molecules, referred to as lung cancer screen 1 (LCS-1). siRNAs against SOD1 slowed the growth of LCS-1 sensitive cell lines; conversely, expression of a SOD1 cDNA increased proliferation of H358 cells and reduced sensitivity of these cells to LCS-1. In addition, SOD1 enzymatic activity was inhibited in vitro by LCS-1 and two closely related analogs. These results suggest that SOD1 is an LCS-1-binding protein that may act in concert with mutant proteins, such as EGFR and KRAS, to promote cell growth, providing a therapeutic target for compounds like LCS-1.

Fig. 1.

Identification of superoxide dismutase 1 (SOD1) as a likely target of LCS-1. (A) Two biologically active 2-phenylpyridazin-3(2H)-one analogs (LCS-1 and LCS-1.28) were immobilized to sepharose 6B beads (closed circles) and used to isolate binding partners in cell extracts of H358 and H1975 cells. (B) The numbers of proteins eluted with unbound compounds (0.5 mM) from the LCS-1 and LCS-1.28 columns and identified by LC-MS/MS. Affinity chromatography and MS identification were performed at least three times for each cell line. (C) The 34 proteins that were eluted from the LCS-1 and LCS-1.28 affinity columns, but not from the gefitinib column were considered candidate targets of LCS-1. (D) H358 and H1975 cells were treated with 0.25 μM LCS-1.34 for 3 h to generate expression signatures and the number of LCS-1.34–regulated genes is shown for each cell line. The 262 genes that were regulated in both H358 and H1975 comprise the LCS-1 gene signature. (E) “Heatmaps” of the altered expression of the 262 LCS-1 gene signature in H358 (Left) and H1975 (Right) cells. The ratio (LCS-1/control) of the average expression of each gene in three replicates is shown in the last column of the heatmaps.

Fig. 2.

SiRNAs to SOD1 reduce the growth of cell lines relatively sensitive to LCS-1. (A) Cell lines that were relatively sensitive (H358 and H1975) or relatively insensitive (A549 and H460) to LCS-1 were transfected with pools of four of the indicated siRNAs. Twenty-four hours after transfection, wells with nontargeting control siRNAs (CONT) were also treated with 0.5 μM LCS-1.34. (B) H358 cells were transfected with the indicated individual siRNAs and 96 h later images of Hoechst-stained cells were taken with an IN Cell Analyzer 2000 wide-field epifluorescence microscope and the total number of Hoechst-labeled nuclei in nine fields per well counted using the object recognition function of Developer 1.7 software. Results represent the average ± SD of two to five experiments in which each condition was assayed in triplicate. In A, the results obtained with each siRNA are expressed relative to the control transfection (CONT) for the respective cell line. (C) H358 cells were transfected with the same siRNAs as in B for 48 h, then extracts prepared and immunoblotted for SOD1 or ENO1 (loading control).

Fig. 3.

Expression of SOD1 correlates inversely with sensitivity to LCS-1 and promotes growth. (A) The IC₅₀ values shown in Table S1 for inhibition of growth of 18 human adenocarcinoma cell lines by LCS-1 are plotted as a function of SOD1 mRNA levels. Spearman's correlation test determined that there was a significant correlation between SOD1 levels and sensitivity to LCS-1 (Spearman's r = 0.6270, P = 0.0054). (B) H358 cells were transfected with a FLAG-tagged SOD1 CDNA or pCMV6 empty vector and G418-resistant populations were selected. Cell extracts were immunoblotted with anti-FLAG (Top), anti-SOD1 (middle) or antitubulin-α (Bottom). (C) Equal number of H358 cells stably expressing SOD1 (H358-SOD1) or control vector (H358-pCMV6) were plated and cell numbers determined daily, starting at day 2. Results represent the mean ± SD of two experiments (performed several months apart) in which each condition was assayed in duplicate. (D) ³H-thymidine incorporation and caspase-3/7 activity were determined 24 h after plating equal numbers of the indicated cell lines as described in SI Materials and Methods. Results represent the mean ± SD of two experiments in which each condition was assayed in triplicate. (E–I) H358-SOD1 (open circle) or H358-pCMV6 (solid square) cells were plated in medium containing increasing concentrations of the indicated compounds; then the numbers of viable cells remaining after 72 h were assessed using AlamarBlue viability dye. Results represent the mean ± SD of at least two experiments in which each condition was assayed in duplicate.

Fig. 4.

Active LCS-1 analogs inhibit SOD1 enzymatic activity in vitro. (A and B) SOD1 enzymatic activity was assayed in vitro in the presence of increasing concentrations of (A) triethylenetetramine tetrahydrochloride or (B) LCS-1 analogs. Note that LCS-1.11 does not inhibit the growth of lung cancer cells. Results represent the mean ± SE of at least three experiments in which each condition was assayed in duplicate.