Loss of ARID1A expression leads to sensitivity to ROS-inducing agent elesclomol in gynecologic cancer cells

Abstract

Inactivating mutations in ARID1A are found in a broad spectrum of cancer types, with the highest frequency in gynecologic cancers. However, therapeutic strategies targeting ARID1A-mutant cancer cells remain limited. In this study, we aimed to identify drugs sensitivities in ARID1A-mutant cancer cell lines. By analyzing the Genomics of Drug Sensitivity in Cancer database, we found that ARID1A-mutant cancer cell lines were more sensitive to treatment with the reactive oxygen species (ROS)-inducing agent elesclomol. In a panel of 14 gynecologic cancer cell lines, treatment with elesclomol inhibited growth and induced apoptosis more potently in ARID1A-mutant cells. Knockdown of ARID1A in RMG1 and OVCA432 ovarian cancer cells resulted in increased sensitivity to elesclomol, whereas restoration of ARID1A expression in TOV21G ovarian cancer cells resulted in increased resistance to elesclomol. Furthermore, we found that knockdown of ARID1A expression resulted in increased intracellular ROS levels. In ovarian clear cell carcinoma patient samples, low expression of ARID1A correlated with high expression of 8-hydroxyguanosine, a marker for oxidative stress. In summary, we demonstrate for the first time that loss of ARID1A leads to accumulation of ROS and suggest that elesclomol may be used to target ARID1A-mutant gynecologic cancer cells.

Keywords: ARID1A; drug sensitivity; elesclomol; ovarian cancer; reactive oxygen species.

Figure 1. ARID1A-mutant cancer cell lines are more sensitive to treatment with the ROS-inducing agent elesclomol than ARID1A-wildtype cells

a. Western blot analysis of ARID1A protein expression in a panel of 14 endometrial and ovarian cancer cell lines. b. Cell growth of endometrial and ovarian cancer cell lines treated with elesclomol for 72 h as measured using the WST-1 assay. Cell growth was quantified relative to DMSO treated controls. c. IC₅₀ values of elesclomol in the cell lines in b. d. Apoptosis of ARID1A-mutant and ARID1A-wildtype cells treated with elesclomol for 72 h as measured using annexin-V and PI staining.

Figure 2. Treatment with elesclomol inhibits cancer cell growth and induces apoptosis by increasing ROS levels

a. Cell growth of SMOV2, IGROV1, and OVCA432 ovarian cancer cells treated with elesclomol in the presence or absence of the antioxidant NAC for 72 h. Cell growth was measured using the WST-1 assay and quantified relative to DMSO treated controls. b. Apoptosis of SMOV2, IGROV1, and OVCA432 ovarian cancer cells treated with elesclomol (ele) for 72 h in the presence or absence of NAC for 72 h as measured using annexin-V and PI staining. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Knockdown of ARID1A expression in ARID1A-wildtype ovarian cancer cells results in increased sensitivity to treatment with elesclomol

a. Cell growth of ARID1A-wildtype RMG1 and OVCA432 cells transfected with ARID1A and non-target siRNA for 24 h and treated with elesclomol for 72 h. b. Cell growth of RMG1 cells after transfection with BRG1, SNF5, and non-target siRNA and treatment as in a. c. Western blot analysis of RMG1 and OVCA432 cells after transfection with ARID1A, BRG1, SNF5, and non-target siRNA for 48 h. Cell growth was measured using the WST-1 assay and quantified relative to DMSO treated controls. *P < 0.05.

Figure 4. Re-expression of ARID1A in ARID1A-mutant ovarian cancer cells results in increased resistance to treatment with elesclomol

a. Cell growth of ARID1A-mutant TOV21G cells after transfection with the pCI-neo-ARID1A and pCI-neo control vectors for 48 h and treatment with elesclomol for 72 h. Cell growth was measured using the WST-1 assay and quantified relative to DMSO treated controls. b. Apoptosis of TOV21G cells after transfection and treatment as described in a as measured using annexin-V and PI staining. c. Western blot analysis showing the re-expression of ARID1A in TOV21G cells. *P < 0.05; **P < 0.01.

Figure 5. Knockdown of ARID1A expression in ARID1A-wildtype ovarian cancer cells results in increased intracellular ROS levels and cell growth

a. Measurement of ROS levels in ARID1A-wildtype RMG1 and OVCA432 cells transfected with ARID1A and non-target siRNA for 72 h using DCFDA. b. Cell growth of RMG1 and OVCA432 cells after transfection with ARID1A and non-target siRNA for 24 h and treatment with the antioxidant NAC for 72 h. Cell growth was measured using the WST-1 assay and quantified relative to DMSO treated non-target control. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Ovarian clear cell carcinoma patient samples with low expression of ARID1A exhibit increased oxidative stress

Expression of ARID1A and 8OHdG were determined using immunohistochemistry. Robust nuclear staining of ARID1A was observed for samples 1-4. Robust cytoplasmic and nuclear staining of 8OHdG was observed for samples 5-8. Photos were taken at 100×.