Synthetic lethality between androgen receptor signalling and the PARP pathway in prostate cancer

Abstract

Emerging data demonstrate homologous recombination (HR) defects in castration-resistant prostate cancers, rendering these tumours sensitive to PARP inhibition. Here we demonstrate a direct requirement for the androgen receptor (AR) to maintain HR gene expression and HR activity in prostate cancer. We show that PARP-mediated repair pathways are upregulated in prostate cancer following androgen-deprivation therapy (ADT). Furthermore, upregulation of PARP activity is essential for the survival of prostate cancer cells and we demonstrate a synthetic lethality between ADT and PARP inhibition in vivo. Our data suggest that ADT can functionally impair HR prior to the development of castration resistance and that, this potentially could be exploited therapeutically using PARP inhibitors in combination with androgen-deprivation therapy upfront in advanced or high-risk prostate cancer.Tumours with homologous recombination (HR) defects become sensitive to PARPi. Here, the authors show that androgen receptor (AR) regulates HR and AR inhibition activates the PARP pathway in vivo, thus inhibition of both AR and PARP is required for effective treatment of high risk prostate cancer.

Fig. 1

AR signalling regulates homologous recombination (HR) and the DNA damage response. a Heat map showing expression of homologous recombination regulators following AR RNAi knockdown, AR transcript and known AR targets/controls are shown mined from the microarray data from LNCaP cells. b Time course of formation of RAD51 foci showing high content cytometry-based quantification of RAD51 nuclear foci number per nucleus in ‘low AR; and ‘high AR’ C4-2 cells in response to 10 Gy radiation. Statistical significance was determined using the Holm–Sidak method, with alpha = 5.000% (n = 3). Scale bar = 1 μm. c Quantification of gene conversion assay from analysis of GFP-positive C4-2-DRGFP cells transfected with non-targeting control siRNA (siNT) or siAR along with ISce1 endonuclease for 72 h followed by detection of GFP-positive cells by flow cytometry. P-value by two-sided Student’s t-test (n = 3); the data represent mean ± SEM. d Foci analysis time course showing high content cytometry-based quantification of the number of γH2AX foci in ‘low AR’ and ‘high AR’ C4-2 cell nuclei in response to radiation (10 Gy). Statistical significance determined using the Holm–Sidak method, with alpha = 5.000%. (n = 3). Scale bar = 1 μm. e High content cytometry analysis of MRN foci in ‘high AR’ and ‘low AR’ C4-2 cells treated with hydroxyurea (HU). Upper panel shows ‘high AR’ and ‘low AR’ nuclei with MRN foci (red dots), lower panel shows a histogram with means of the errors; P-value by two-sided Student’s t-test (n = 2). f Micrographs from biopsies showing examples of Ki67- and RAD51 positive cells and Ki67 positive alone. Scale bar = 5 μm. g Graph showing percentage of RAD51-positive cells in the population of Ki67-positive cells in biopsies after indicated treatment. Error bars shows SEM and ** = P < 0.01, Mann–Whitney test

Fig. 2

Androgen deprivation therapy triggers PARP activation. a Western blot showing C4-2 cells treated for 72 h with indicated agents (10 µM). b Western blot showing C4-2 cells transfected with indicated siRNA (20 nM). c Immunofluorescence microscopy images showing relative intensities of PAR Poly(ADP Ribose) and PARP1 (Poly ADP-ribose polymerase 1) in PCa tissue pre- (pre-castration or AR+) and post leuprolide (post-castration or AR-) treatment (8 weeks). Scale bar = 100 μm. d Histogram shows PARP activity in PCa patients pre- and post-leuprolide treatment (One sample t-test, two-tailed, test value 100)

Fig. 3

Synthetic lethality between AR and PARP pathways in PCa. a, b Viable fraction assessed by MTS assay, of C4-2 (a) and (b) LN3 cells treated with the indicated doses of Olaparib and/or enzalutamide or bicalutamide (10 μM) for 7 days or until 95% confluence was reached; P-value by two-sided Student’s t-test; bars show mean ± SEM. c Live cell imaging confluence analysis (Incucyte) of C4-2 cells treated with Olaparib (1 μM), enzalutamide (10 μM), bicalutamide (10 μM) or combined treatment, statistical significance calculated by two-way ANOVA. d Live cell imaging confluence analysis (Incucyte) of ‘high AR’ and ‘low AR’ C4-2 cells treated with doxycycline and/or Olaparib (1 μM), statistical significance calculated using two-way ANOVA. e, f Clonogenic survival assay for C4-2 cells with either inducible shNT (e) or shAR (f); cells were treated with doxycycline and 1 μM Olaparib, P-value by two-sided Student’s t-test; bars show mean ± SEM. All experiments were independently performed in triplicates. The data represent means ± SEM. P-values from significant two-sided Student’s t-tests are given (* = P < 0.05)

Fig. 4

Dual inhibition of AR and PARP1/2 function represses PCa growth. a Tumour xenografts of C4-2 cells. NSG mice were treated with DMSO (vehicle) or bicalutamide and/or Olaparib for 6 weeks, statistical significance calculated using two-way ANOVA. b Tumour xenografts of PC3-ctl or PC3-AR cells, NSG mice were administered with DMSO (vehicle) or Olaparib as indicated, P-value by two-sided Student’s t-test. c, d Quantification of ki67 expression in ex vivo culture of human PCa treated with (c) bicalutamide (10 μM) or (d) Enzalutamide (10 μM) and/or Olaparib (2 μM) for 72 h, significance calculated by Mann–Whitney U test