High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: interaction with IQ motif-containing factors

Abstract

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC50 for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA(+)/CD24(-/low)/CD44(+) cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.

Keywords: IQGAP1; MYH9; cancer stem cells; disulfiram; high-throughput screens; triple-negative breast cancer.

Figures

Figure 1. High-throughput screen of 3185 compounds with known biological activities against 4 human-derived TNBC cell lines (MDA-MB-231, MDA-MB-436, HCC70, Bt549). Shown are the average responses by the 4 lines to (A) Spectrum library (1 µM, 2000 drugs), (B) Prestwick library (0.8 µM, 1185 drugs). (C and D) Top 5 hits from the Spectrum and Prestwick libraries; disulfiram and thiram are highlighted in red. Values represent the average cell viability of all 4 lines expressed as a percentage of vehicle treated control. (E–J) Validation and dose-response curves for select hits using alamar blue viability assay, performed in triplicate.

Figure 2. Dose-response curves for a panel of 13 human-derived TNBC cell lines treated with disulfiram or thiram. (A) Response to disulfiram for each individual line by MTT viability assay. Average IC50 = 300 nM. n = 3–5, each performed in triplicate. (B) Response to thiram for each individual line. Average IC50 = 360 nM. n = 3–5, each performed in triplicate. (C) Average response to disulfiram based on TNBC subtype. (D) Average response to thiram based on TNBC subtype. Basal-like (BaA): HCC1954, HCC1569, HCC3153, HCC70, HCC1937, and MDA-MB-468. Claudin-low (BaB): MDA-MB-436, MDA-MB-231, MDA-MB-157, Bt549, SUM149, Hs578t, and HCC38.

Figure 3. Disulfiram binds IQGAP1 and MYH9 to partially inhibit cell growth. (A) DARTS was performed on MDA-MB-231 cell lysates incubated with either vehicle (DMSO) or varying concentrations of disulfiram and visualized via silver staining. Mass spectrometry analysis of disulfiram-protected bands identified IQGAP1 (red arrowhead) and MYH9 (black arrowheads). (B) DARTS–western blot analysis was performed to validate IQGAP1 and MYH9 as binding targets of disulfiram. β-actin was used as a negative control. IQGAP1 and MYH9 over β-actin ratios, calculated from ImageJ analysis, are shown. Arrow indicates band used to calculate enrichment ratio over control. (C) DARTS–western blot analysis using BT-549 (Basal B) cell lysates. GAPDH was used as a negative control. (D and E) western blot analysis demonstrating efficient knockdown of IQGAP1 and MYH9 after treatment with RNAi relative to disulfiram (DSF), DharmaFECT 4 transfection reagent, or no treatment. (F) MTT viability assay after treatment with MYH9 or IQGAP1 siRNA, or disulfiram (DSF). Values represent % of vehicle control. n = 3, each performed in triplicate.

Figure 4. Pathway analysis of MDA-MB-231 cells 72 h post treatment with 100 nM or 250 nM disulfiram (DSF), compared with untreated control. Expression data were analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID) and visualized by “functional enrichment maps”. Pathways of interest are marked with red boxes.

Figure 5. Effect of combination treatment with disulfiram (DSF) and doxorubicin (Dox). (A) MDA-MB-231 cells treated with DSF and Dox in combination or alone and analyzed by MTT assay. (B) MDA-MB-468, HCC38, HCC70, and Hs578t treated with their respective IC50 doses of DSF and Dox in combination or alone. All values represent % of untreated control. † denotes additive effect; ‡ denotes synergistic effect. (See “Materials and Methods”).

Figure 6. Apoptosis and senescence analysis of MDA-MB-231 cells treated with disulfiram and/or doxorubicin. Cells were treated with disulfiram and/or doxorubicin at 250 nM and 125 nM, respectively. (A) Flow cytometry profiles of 7-AAD and Annexin-V for each experimental condition at 24 h and 72 h post treatment. 7-AAD−/Annexin-V+ marks early stage apoptosis, 7-AAD+/Annexin-V+ late stage apoptosis, and 7-AAD+/Annexin-V− necrotic cells. (B) Percent senescence in untreated and treated MDA-MB-231 cells, and MCF7-positive control (doxorubicin 125 nM), as determined by senescence-associated β-galactosidase (X-gal/BCIG) staining. (C–G) Light microscope view of MDA-MB-231 cells treated with (C) vehicle control, (D) doxorubicin, (E) disulfiram, or (F) doxorubicin plus disulfiram, and (G) MCF7 positive control (doxorubicin 125 nM). Scale bars represent 200 µm.

Figure 7. Effect of disulfiram and doxorubicin on the CSC fraction in MDA-MB-231 cells. (A and B) Gating conditions for live (7-AAD negative) and ESA+ cells. (C–E) Effect of disulfiram (250 nM) and doxorubicin (125 nM) on the CD44+/CD24-/low/ESA+ cancer stem cell (CSC) fraction (red box). (F) Average absolute values and normalized ratios of CSC fraction.