Targeted blockade of HSP90 impairs DNA-damage response proteins and increases the sensitivity of ovarian carcinoma cells to PARP inhibition

Abstract

Pharmacological inhibition of PARP is a promising approach in treating high grade serous ovarian carcinoma (HGSOC). PARP inhibitors (PARPi) are most active in patients with defects in DNA damage repair (DDR) mechanisms, such as alterations in expression/function of DNA repair and homologous recombination (HR) genes/proteins, including BRCA1 and BRCA2. Benefit of PARPi could be extended towards HR-proficient patients by combining PARPi with agents that functionally abrogate HR. An attractive molecular target for this purpose is heat shock protein 90 (HSP90), which mediates the maturation and stability of several key proteins required for DDR. Here, we tested the hypothesis that targeted inhibition of HSP90 with a small-molecule inhibitor ganetespib would sensitize non-BRCA mutant ovarian carcinoma (OC) cells to PARP inhibition by talazoparib. We used commercially available cell lines, along with several novel HGSOC OC cell lines established in our laboratory. Ganetespib treatment destabilized HSP90 client proteins involved in DNA damage response and cell cycle checkpoint, and disrupted γ-irradiation-induced DNA repair. The effects of the combination of ganetespib and talazoparib on OC cell viability and survival were also analyzed, and among the non-BRCA mutant cell lines analyzed, the combination was synergistic in several cell lines (OVCAR-3, OC-1, OC-16). Together, our data suggest that ganetespib-mediated inhibition of HSP90 effectively disrupts critical DDR pathway proteins and may sensitize OC cells without 'BRCAness' to PARPi. From a clinical perspective, this suggests that HSP90 inhibition has the potential to sensitize some HGSOC patients without HR pathway alterations to PARPi, and potentially other DNA-damage inducing agents.

Keywords: HSP90 inhibitor; Ovarian cancer; PARP inhibitor; targeted therapy.

Figure 1.

Ganetespib destabilizes DNA repair and cell cycle checkpoint proteins in ovarian carcinoma cells in a dose- and time-dependent manner. OVCAR-3, OC-1 and OC-38 cells were treated with 0 (vehicle), 12.5, 25, 50, 100, or 500 nM/L of ganetespib for 24 h (A), or with 50 nM/L ganetespib for 0, 6, 12, 24, 36, and 48 h (B). Cells were then lysed and subjected to immunoblot analysis with antibodies against BRCA2, BRCA1, HSP90, MRE11, c-MYC, CDK1, CHK1, ATM, and RAD51. Detection of GAPDH levels was used as a loading control.

Figure 2.

Ganetespib treatment disrupted ionizing radiation-induced DNA-repair and homologous recombination. OVCAR-3, OC-1, and OC-38 cells were treated with vehicle or 25 nM/L ganetespib for 24 h before IR exposure and analyzed by immunofluorescent detection of γ-H2AX⁺ or RAD51⁺ foci. (A) Representative images of control and irradiated OVCAR-3, OC-1, and OC38 cells 8 h post-IR (scale bar = 20 µm). (B) Quantification of the number of γ-H2AX foci per nucleus and percentage of cells with RAD51⁺ foci. (C) Representative images 8 h post-IR (scale bar = 20 µm) and quantification of pATM^S1981 positive foci in OVCAR-3 cells treated with 25 nM/L ganetespib for 24 h before IR exposure. Data were analyzed by One-way Analysis of Variance (ANOVA) and the non-parametric Kruskal–Wallis test followed by Dunns post-test. For all analyses, P values <0.05 were considered significant (*P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 3.

Ganetespib sensitizes ovarian carcinoma cells to talazoparib. Viability curves of OVCAR-3 (A), UWB 1.289 (B), OC-1 (C), OC-16 (D) cells treated with ganetespib and talazoparib alone or in combination at the indicated non-fixed concentrations or fixed molar ratios. Viability curves of OVCAR-3 cells treated with ganetespib in combination with rucaparib (E) and niraparib (F) in 1:1 molar ratio. Only statistically significant (P values <0.05) synergistic PARPi and ganetespib combinations are shown.

Figure 4.

Combined treatment with ganetespib and talazoparib significantly increases cell death in HGSOC cells. OVCAR-3 (A) and OC-1 (B) cells treated with vehicle, talazoparib, ganetespib, or talazoparib + ganetespib and analyzed for induction of cell death by Annexin V and 7-AAD staining and increased levels of cleaved PARP. Representative flow cytometry dot plots of Annexin V and 7AAD stained cells are shown with quantification of the Annexin V⁺ cells and immunoblot detection of cleaved PARP and GAPDH (loading control).