The combined effect of USP7 inhibitors and PARP inhibitors in hormone-sensitive and castration-resistant prostate cancer cells

Abstract

Purpose of the study: Reduced levels of the tumor suppressor protein CCDC6 sensitize cancer cells to the treatment with PARP-inhibitors. The turnover of CCDC6 protein is regulated by the de-ubiquitinase USP7, which also controls the androgen receptor (AR) stability. Here, we correlated the expression levels of CCDC6 and USP7 proteins in primary prostate cancers (PC). Moreover, we tested the efficacy of the USP7 inhibitors, in combination with PARP-inhibitors as a novel therapeutic option in advanced prostate cancer.Experimental techniques: PC cells were exposed to USP7 inhibitor, P5091, together with cycloheximide, to investigate the turnover of the USP7 substrates, AR and CCDC6. As outcome of the AR downregulation, transcription targets of AR and its variant V7 were examined by qPCR. As a result of CCDC6 degradation, the induction of PARP inhibitors sensitivity was evaluated by analyzing PC cells viability and foci formation. We scored and correlated CCDC6 and USP7 expression levels in a prostate cancer tissue microarray (TMA).

Results: P5091 accelerated the degradation of AR and V7 isoform affecting PSA, UBE2C, CDC20 transcription and PC cells proliferation. Moreover, P5091 accelerated the degradation of CCDC6 sensitizing the cells to PARP-inhibitors, that acted sinergistically with genotoxic agents. The immunohistochemical analysis of both CCDC6 and USP7 proteins exhibited significant correlation for the intensity of staining (p ≤ 0.05).Data interpretation: Thus, CCDC6 and USP7 represent predictive markers for the combined treatment of the USP7-inhibitors and PARP-inhibitors in advanced prostate cancer.

Keywords: ARFL and V7; CCDC6; P5091; USP7; olaparib.

Figure 1. Pharmacological inhibition of USP7 affects prostate cancer cell proliferation

(A) Immunoblot analysis of USP7, CCDC6, AR and ARV7 isoform in human LNCaP, 22RV1 and PC3 prostate cancer cell lines. Antitubulin is shown as loading control. (B) LNCaP cells were treated with vehicle or different concentrations of P5091, as indicated, and cells were counted at the indicated times, in the presence or absence of DHT (10 nM). (C) PC3 cells were treated with vehicle or different concentration of P5091, as indicated, and cells were counted at the indicated times. In B and C the values are the mean +/− SD of three independent experiments. (D) USP7 inhibitors P5091 shows dose-dependent cytotoxic effect in prostate cancer cell lines. Cells were seeded in 96-well plates and 24 h later exposed to vehicle or P5091 at the indicated doses, in presence or absence of Z-VAD-FMK (20 μM), for 144 h and analysed for viability using a modifeid 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay. CellTiter 96 Aqueous One Solution assay (Promega), as 50% inhibitory concentration (IC50) values. The value are presented as mean standard deviation of three independent experiments. Surviving fraction of LNCaP and PC3 cells are shown. (E) Caspase 3 activity was evaluated in LNCaP and PC3 cells treated or not treated with P5091 for 24 h, as indicated. The plotted values represent the mean +/− s.e.m. of three independent experiments.

Figure 2. The USP7 inhibitor P5091 shows antiproliferative effects, affects CCDC6, AR and V7-isoform half lives and impairs androgen-responsive genes expression in 22Rv1 cells

(A) 22Rv1 cells were treated with vehicle or different concentrations of P5091, as indicated, and cells were counted at the indicated times, in the presence or absence of DHT (10 nM). The values are the mean +/− SD of three independent experiments. (B) USP7 inhibitors P5091 shows dose-dependent cytotoxic effect in prostate cancer cells. Cells were seeded in 96-well plates and 24 h later exposed to vehicle or P5091 at the indicated doses, in presence or absence of Z-VAD-FMK (20 μM), for 144 h and analysed for viability using a modifeid 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay. CellTiter 96 Aqueous One Solution assay (Promega), as 50% inhibitory concentration (IC50) values. The value are presented as mean standard deviation of three independent experiments. Surviving fraction of 22Rv1 cells is shown. (C) Caspase 3 activity was evaluated in 22Rv1 cells treated or not treated with P5091 for 24 h, as indicated. The plotted values represent the mean +/− s.e.m. of three independent experiments. (D) 22Rv1 cells were pretreated with either vehicle or P5091 (6 μM, IC50 in 22Rv1 cells), for 4 h, followed by the addition of cycloheximide (CHX) at 50 μg/ml for the indicated times. Total proteins lysates were subjected to immunoblot analysis using anti-CCDC6, anti-AR (able to detect the full lenght and the V7 isoform) or anti-PCNA antibodies. (E) Expression of AR-target genes levels in 22Rv1 cells was determined by qPCR, following vehicle or P5091 treatment (25 μM) for 24 h, and normalized against expression of GAPDH. (F) Expression of ARV7-target genes levels in 22Rv1 cells was determined by qPCR, following vehicle or P5091 treatment (25 μM) for 24 h, and normalized against expression of GAPDH. In C and D the values are the mean +/− SD of three independent experiments.

Figure 3. P5091 controls CCDC6 stability and affects the DSBs DNA repair in prostate cancer cells

(A) LNCaP and (B) PC3 prostate cancer cells were pretreated with either vehicle or P5091 (6 μM IC50 in LNCaP and PC3) for 4 h, followed by the addition of cycloheximide (CHX) at 50 μg/ml for the indicated times. Total proteins lysates were subjected to immunoblot analysis using anti-CCDC6 or anti-PCNA antibodies. Anti-AR antibody is also shown in the LNCaP cells. (C) LNCaP and PC3 cells were transfected with DR-GFP alone, as control, or together with I-SceI. The percentage of GFP positive cells, compared to controls, have been plotted as histograms that are representative of three independent experiments. Error bars indicate the standard error mean. (D) LNCaP and PC3 cells were exposed to 5Gy IR, in presence or not of 5 μM of P5091, followed by 18 h recovery. Immunofluorescence images of the cells stained for Rad51 are shown. Immunofluorescence of LNCaP and PC3 upon P5091 treatment only are also shown. Cells containing more than five foci were scored as positive. The percentage of Rad51 positive nuclei at 18 h from irradiation are shown on the right of the images. Error bars represent standard error mean. Results are representative of at least two independent experiments.

Figure 4. The USP7 inhibitor P5091 sensitizes the prostate cancer cells to PARP-inhibitors

(A) Left: Surviving fractions of PC3 cells treated, in presence or absence of P5091 (2.5 μM), with olaparib at the indicated doses for 144 h are shown. Right: drugs sensitivity to olaparib and etoposide, in presence or absence of P5091 (2.5 μM) was determined by a modified 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay, CellTiter 96 Aqueous One Solution assay (Promega), as 50% inhibitory concentration (IC50) values. CI according to 1:2 concentration ratio of etoposide and olaparib, in presence or absence of P5091 (2.5 μM), are shown. (B) Top: Surviving fractions of LNCaP cells treated, in presence or absence of P5091 (2.5 μM), with olaparib at the indicated doses for 144 h are shown. DHT (10 nM) was added as indicated (− / +). Bottom: drugs sensitivity to olaparib and to etoposide in presence or absence of P5091 (2.5 μM) was determined by a modified 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay, CellTiter 96 Aqueous One Solution assay (Promega), as 50% inhibitory concentration (IC50) values. CI according to 1:2 concentration ratio of etoposide and olaparib, in presence or absence of P5091 (2.5 μM), are shown. CI < 1, CI = 1, CI > 1 indicate synergism, additive effect and antagonism, respectively. In A, on the right, and in B, at the bottom, the values are presented as mean standard deviation of three independent experiments.

Figure 5. Combined effect of USP7 inhibitors and PARP inhibitors in the 22Rv1 cells

(A) and (B) Top: Surviving fractions of 22Rv1 cells treated, in presence or absence of P5091 (2.5 μM), with olaparib at th indicated doses for 144 h are shown. DHT (10 nM) was added as indicated (−/+). Bottom: Drugs sensitivity to olaparib in presence or absence of P5091 (2.5 μM) was determined by a modified 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay, CellTiter 96 Aqueous One Solution assay (Promega), as 50% inhibitory concentration (IC50) values.

Figure 6. Highly concordant expression of CCDC6 and USP7

(A–D) Two representative cases of prostate adenocarcinoma (A, B case1; C, D case 2): (A, C) CCDC6 stain (100×), respectively high expression (A) and low expression (C); (B, D) USP7 stain (100×), respectively high expression (B) and low expression (D); (E) Scatter plot showing the relationship between USP7 and CCDC6 IHC expression as from prostate adenocarcinoma TMA analysis (correlation coefficient R = 0.855); (F) The table summarizes the weighted kappa and the Spearman's rank correlation analyses results, both proved to be extremely significant.