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, 133 (3), 599-606

Suberoylanilide Hydroxamic Acid (SAHA) Enhances Olaparib Activity by Targeting Homologous Recombination DNA Repair in Ovarian Cancer

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Suberoylanilide Hydroxamic Acid (SAHA) Enhances Olaparib Activity by Targeting Homologous Recombination DNA Repair in Ovarian Cancer

Panagiotis A Konstantinopoulos et al. Gynecol Oncol.

Abstract

Objectives: Approximately 50% of serous epithelial ovarian cancers (EOC) contain molecular defects in homologous recombination (HR) DNA repair pathways. Poly(ADP-ribose) polymerase inhibitors (PARPi) have efficacy in HR-deficient, but not in HR-proficient, EOC tumors as a single agent. Our goal was to determine whether the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize HR-proficient ovarian cancer cells to the PARPi AZD-2281 (olaparib).

Methods: Ovarian cancer cell lines (SKOV-3, OVCAR-8, NCI/ADR-Res, UWB1.289 BRCA1null and UWB1.289+BRCA1 wild-type) were treated with saline vehicle, olaparib, SAHA or olaparib/SAHA. Sulforhodamine B (SRB) assessed cytotoxicity and immunofluorescence and Western blot assays assessed markers of apoptosis (cleaved PARP) and DNA damage (pH2AX and RAD51). Drug effects were also tested in SKOV-3 xenografts in Nude mice. Affymetrix microarray experiments were performed in vehicle and SAHA-treated SKOV-3 cells.

Results: In a microarray analysis, SAHA induced coordinated down-regulation of HR pathway genes, including RAD51 and BRCA1. Nuclear co-expression of RAD51 and pH2AX, a marker of efficient HR repair, was reduced approximately 40% by SAHA treatment alone and combined with olaparib. SAHA combined with olaparib induced apoptosis and pH2AX expression to a greater extent than either drug alone. Olaparib reduced cell viability at increasing concentrations and SAHA enhanced these effects in 4 of 5 cell lines, including BRCA1 null and wild-type cells, in vitro and in SKOV-3 xenografts in vivo.

Conclusions: These results provide preclinical rationale for targeting DNA damage response pathways by combining small molecule PARPi with HDACi as a mechanism for reducing HR efficiency in ovarian cancer.

Keywords: Histone deacetylase inhibitors (HDACi); Olaparib (Ola); Ovarian cancer; Phosphorylated gamma histone H2AX (pH2AX); Poly(ADP-ribose) polymerase inhibitors (PARPi); Vorinostat (SAHA).

Conflict of interest statement

CONFLICT OF INTEREST

The authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1. SAHA down-regulates Homologous Recombination (HR) pathway genes in SKOV-3 cells
A) A heat map expression plot of 15 downregulated HR pathway genes in SKOV-3 cells after 24 h treatment with 1μM SAHA compared to vehicle (0.01% DMSO) treated controls. B) A representative Western blot showing 1μM SAHA-induced downregulation of RAD51 and BRCA1 expression after 24 h treatment. Actin was used as a loading control.
Figure 2
Figure 2. SAHA decreases formation of RAD51 foci in SKOV-3 cells in vitro
Cells pre-treated with 0.5μM cisplatin for 6 h were then treated with vehicle (0.01% DMSO), olaparib (OLA) (10μM), SAHA (1μM) or the combination for a further 24 h. A) Representative images (40X) of IF staining for RAD51 (green) and pH2AX (red). DAPI-stained nuclei are in blue. B) The percentage of cells showing co-expression of RAD51 and pH2AX (>5 foci was considered positive staining) was calculated for each drug. Values are mean + SD for 3 independent experiments. At least 100 cells were counted (x40) for each drug treatment per experiment. * p < 0.01 compared to vehicle; b p < 0.01 relative to olaparib alone, both Student’s t test, Student’s t test.
Figure 3
Figure 3. Olaparib combined with SAHA increases SKOV-3 cell apoptosis and expression of the DNA damage mark pH2AX in vitro
SKOV-3 ovarian cancer cells were treated with vehicle (0.01% DMSO), olaparib (OLA) (10μM), SAHA (1μM) or the combination for 24 h and evaluated by Western blot and IF staining for the expression of the DNA damage marker, pH2AX, and the apoptotic marks, cleaved PARP and cleaved caspase-3. A) IF nuclear stain for pH2AX (green). DAPI-stained nuclei are in blue. The number of cell nuclei displaying less than 6 foci (negative), between 6–20 foci, greater than 20 foci and diffuse pan-nuclear (Pan-nuc) staining for pH2AX foci was quantified and is presented in percentages (40X). B) Representative Western blot analysis of cleaved PARP and cleaved caspase 3 in whole cell lysates, and pH2AX in histone extracts. Loading controls were β-actin and histone H3, respectively. C) The percentage of cells showing cleaved PARP expression was calculated for each drug. D) Representative images for IF stain for cleaved PARP (green). DAPI-stained nuclei are in blue. Values in A) and C) are mean + SD for 5 and 3 independent experiments, respectively. At least 100 cells were counted (40X) for each drug treatment per experiment. * p < 0.01 compared to vehicle; a p < 0.01 relative to SAHA alone; b p < 0.01 relative to olaparib alone, all Student’s t test.
Figure 4
Figure 4. Olaparib reduces cell viability alone and combined with SAHA in ovarian cancer cells
A,C,D) Ovarian cancer cells (SKOV-3, OVCAR-8, NCI/ADR-Res, UWB1.289 BRCA1 null and UWB1.289+BRCA1 wild-type) were treated with olaparib (OLA) (5–100μM), SAHA (0.5–10μM) or both. SRB assays were performed to assess cytotoxicity and cell viability after 72 h of treatment. Each dose was replicated 6 times. Values are shown as the mean + SD for three independent experiments. B) Combination results for the drug treatments are shown. A CI of < 1.0 is considered to be synergistic.
Figure 5
Figure 5. SAHA inhibits tumor growth alone and in combination with OLA in vivo
Nude mice injected SC with SKOV-3 cells were treated with olaparib (OLA) (10mg/kg thrice weekly), SAHA (50mg/kg twice thrice weekly), the OLA/SAHA combination at the same doses and schedule, and vehicle (0.01% DMSO thrice weekly) for 3 weeks following establishment of the tumors (approx 200mm3). 5 mice per treatment group were used. A) Tumor volume change from start of treatment period to sacrifice (%). In harvested tumors, the percentage of cells staining for B) pH2AX, C) cleaved caspase-3 and D) ki67/mib-1 by IHC was calculated. Values in B–D) are mean + SD for 3 independent experiments. At least 1000 cells were counted (40X) for each drug treatment per experiment. * p < 0.02 compared to vehicle; a p < 0.02 relative to SAHA alone; b p < 0.01 relative to olaparib alone, Mann-Whitney t test. E) High power (x40) images of H&E, pH2AX, cleaved caspase-3 and ki67/mib-1 staining in representative tumor sections for each drug treatment.

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