Targeting Plk1 to Enhance Efficacy of Olaparib in Castration-Resistant Prostate Cancer

Abstract

Olaparib is an FDA-approved PARP inhibitor (PARPi) that has shown promise as a synthetic lethal treatment approach for BRCA-mutant castration-resistant prostate cancer (CRPC) in clinical use. However, emerging data have also shown that even BRCA-mutant cells may be resistant to PARPi. The mechanistic basis for these drug resistances is poorly understood. Polo-like kinase 1 (Plk1), a critical regulator of many cell-cycle events, is significantly elevated upon castration of mice carrying xenograft prostate tumors. Herein, by combination with Plk1 inhibitor BI2536, we show a robust sensitization of olaparib in 22RV1, a BRCA1-deficient CRPC cell line, as well as in CRPC xenograft tumors. Mechanistically, monotherapy with olaparib results in an override of the G₁-S checkpoint, leading to high expression of Plk1, which attenuates olaparib's overall efficacy. In BRCA1 wild-type C4-2 cells, Plk1 inhibition also significantly increases the efficacy of olaparib in the presence of p53 inhibitor. Collectively, our findings not only implicate the critical role of Plk1 in PARPi resistance in BRCA-mutant CRPC cells, but also shed new light on the treatment of non-BRCA-mutant patient subgroups who might also respond favorably to PARPi. Mol Cancer Ther; 16(3); 469-79. ©2017 AACR.

Figure 1. Plk1 inhibition enhances the efficacy of Olaparib in BRCA1-deficient 22RV1 cells

A, 22RV1 cells were treated with indicated concentrations of BI2536, Olaparib or both for 24 h and harvested for immunoblotting (IB) with antibodies against cleaved-poly (ADP-ribose) polymerase (PARP). B, 22RV1 cells (0.5 × 10³) were plated in 6-well plates for 24 h and then treated with BI2536 (2.5 nM), Olaparib (1 µM) or both drugs. After changing fresh media containing drug(s) every 3 days for two weeks, cells were PFA fixed and colony formation was monitored by crystal violet staining. The experiments shown are representatives of 3 repeats. C, Quantification of the colonies in (B). The numbers of colonies were quantified using ImageJ software (mean ± SD; n=3 independent experiment). p value = 1.04648E-07 (BI2536 vs BI2536+Olaparib); p value = 9.11184E-06 (Olaparib vs BI2536+Olaparib). **, p < 0.001. D, Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells following BI2536 (5 nM) and Olaparib (10 µM) treatment or combination of both for 24 h in 22RV1 cells. E, Quantification of apoptotic cells (annexin-V positive + PI positive + annexin-V and PI double positive cells). p value = 0.000884 (BI2536 vs BI2536+Olaparib); p value = 0.000985 (Olaparib vs BI2536+Olaparib). **, p value < 0.001). F, Representative images of immunofluorescence (IF) staining for γH2AX in 22RV1 cells treated with of BI2536 (5 nM), Olaparib (10 µM) or both for 24 h. 22RV1 cells (1 × 10⁵) were plated in 6-well plates on day 0 and then treated with BI2536, Olaparib or both for 24 h. G, Quantification of the γH2AX signaling in (F). For quantification, at least 300 cells were scored for γ-H2AX foci and percentages of cells with 5 or more foci are shown. Values are mean ± SEM of three independent experiments. p value = 0.0013512 (BI2536 vs BI2536+Olaparib); p value = 0.009928462 (Olaparib vs BI2536+Olaparib). *, p < 0.05 (two-tailed unpaired t-test). H, 22RV1 cells were treated with BI2536 (5 nM), Olaparib (10 µM) or combination of 2 drugs for 24 h and harvested for IB.

Figure 2. Combination therapy with BI2536 and Olaparib synergistically inhibits growth of 22RV1-derived xenograft tumors

A, Tumor growth curves of the 22RV1-derived xenograft mouse study. Nude mice were inoculated with 22RV1 cells (1×10⁶) for two weeks, intravenously injected with BI2536 (12 mg/kg), Olaparib (50 mg/kg) by oral gavage or a combination of both drugs. The sizes of the tumors in each group were measured every 3 or 4 days (mean ± SEM; n = 3 mice from each experiment group). “NS” represents “no significant difference”; *, p < 0.05 compared with the BI2536 or Olaparib treated monotherapy group or the untreated group at the end of the study. B, Images of the 22RV1-derived xenograft tumors at the end of the study. C, Tumor weight measurement after being freshly removed from the bodies. **, p < 0.001 compared with the BI2536 or Olaparib-treated monotherapy group at the end of the study.

Figure 3. Histological analysis of 22RV1-derived xenograft tumors

A, Representative images of H&E on formaldehyde-fixed, paraffin-embedded 22RV1-derived xenograft tumors sections from different treatment groups. B, Representative images of immunofluorescence staining for Ki67 and cleaved caspase 3 with tumors as in (A). C & D, Microscopic quantification of Ki67 or cleaved caspase 3 as percentages of Ki67- or cleaved caspase 3-positive cells in total numbers of cells. For quantification, at least 300 cells were scored on each field (20× fields, more than 3 sections at different tumor depths per mouse) as the percentages of Ki67 or cleaved-caspase 3 positive cells compared to the total numbers of cells counted. *: p < 0.05, **: p < 0.001 (two-tailed unpaired t-test).

Figure 4. Plk1 inhibition profoundly suppresses the level of PARP1 protein expression and PARP1 activation inhibits the Plk1 expression

A, PARP1 binds to Plk1. 22RV1 cells were treated with nocodazole and harvested for immunoprecipitation (IP) with antibodies against Plk1, followed by IB. B, 22RV1 cells were treated with 0.3 mM mimosine (Mimo) for 20 h, 4 mM hydroxyurea (HU) for 24 h, or 200 ng/ml nocodazole (Noc) for 12 h to block cells at G1, G1/S or G2/M phase, respectively. Cell were harvested after 30 minutes treatment with 50 µM MNNG. C, Representative images of IF staining for PARP1 and Plk1 in 22RV1 cells treated with 0.3 mM mimosine for 20 h, 4 mM hydroxyurea for 24 h, 10 µM RO-3306 for 7 h or 200 ng/ml nocodazole (Noc) for 12 h. D, 22RV1 cells were treated with BI2536 (5 nM, 24 h) or in combination with MNNG (50 µM), and harvested at indicated time points for IB. E, 22RV1 cells were treated with BI2536 (5 nM, 24 h), MNNG (50 µM, 30 minutes), or both, and subjected to FACS analysis.

Figure 5. p53 inhibition restores the synergistic effect between BI2536 and Olaparib in BRCA1 wild type C4-2 cells

A to C, 22RV1 cells were treated with or without Olaparib (10 µM) for 24 h, and harvested for IB (A), quantitative RT-PCR (B) or FACS analysis (C). D, BRCA1 expression was determined by IB in 22RV1 and C4-2. E to G, C4-2 cells were treated with or without Olaparib (10 µM) for 24 h and harvested for IB (D), quantitative RT-PCR (E) or FACS analysis (F). H, C4-2 cells were treated with indicated concentrations of BI2536, Olaparib or both for 24 h and harvested for IB with antibodies against cleaved-PARP. I, C4-2 cells (0.5 × 10³) were plated in 6-well plates for 24 h and treated with BI2536, Olaparib or both drugs. After changing fresh media containing drug(s) every 3 days for two weeks, cells were PFA fixed and colony formation was monitored by crystal violet staining. The experiments shown are representatives of 3 repeats. J, C4-2 cells were treated with BI2536 (5 nM), Olaparib (10 µM) or both drugs in the presence/absence of p53 inhibitor Pifithrin-1α (PFT1-α, 40 µM) and harvested for IB. K to L: PC3 or MDA-MB-231 cells were treated with indicated concentrations of BI2536, Olaparib or both for 24 h and harvested for IB. M, Proposed working model based on the results of this study.