SIRNA-directed in vivo silencing of androgen receptor inhibits the growth of castration-resistant prostate carcinomas

Abstract

Background: Prostate carcinomas are initially dependent on androgens, and castration or androgen antagonists inhibit their growth. After some time though, tumors become resistant and recur with a poor prognosis. The majority of resistant tumors still expresses a functional androgen receptor (AR), frequently amplified or mutated.

Methodology/principal findings: To test the hypothesis that AR is not only expressed, but is still a key therapeutic target in advanced carcinomas, we injected siRNA targeting AR into mice bearing exponentially growing castration-resistant tumors. Quantification of siRNA into tumors and mouse tissues demonstrated their efficient uptake. This uptake silenced AR in the prostate, testes and tumors. AR silencing in tumors strongly inhibited their growth, and importantly, also markedly repressed the VEGF production and angiogenesis.

Conclusions/significance: Our results demonstrate that carcinomas resistant to hormonal manipulations still depend on the expression of the androgen receptor for their development in vivo. The siRNA-directed silencing of AR, which allows targeting overexpressed as well as mutated isoforms, triggers a strong antitumoral and antiangiogenic effect. siRNA-directed silencing of this key gene in advanced and resistant prostate tumors opens promising new therapeutic perspectives and tools.

Figure 1. Silencing of AR in LNCaP cells and tumors.

A: Control (cont)- panAR- or hAR-siRNA were transfected into human LNCaP or into mouse Sertoli TM4 cells. AR was immunodetected by western blot in cell lysates 2 days after removal of transfection medium. α-tubulin (tub) expression was used as a loading control. B: Relative PSA mRNA level in LNCaP cells transfected with control or hAR-siRNA and grown for 48 h in the absence of androgens or in the presence of R1881, 0.5 nM (mean±SE, n = 3 independent experiments). Similar results were obtained using the panAR-siRNA. **p<0.01 as compared to values in the absence of androgens. C: LNCaP cells were subcutaneously injected on day 0 to nude mice. Starting from day 51 (arrow), animals (5 per group) received a daily i.p. injection of 3 µg of cont- (black symbols) or panAR-siRNA (white symbols) diluted in 50 µl saline; tumor volume (cm³, mean±SE, n = 5). *p<0.05 and **p<0.01 comparing panAR-siRNA to cont-siRNA treated tumors. D: Analysis of AR expression by immunohistochemistry (left panels) and apoptotic cells by TUNEL (right panels) in representative tumors collected at the end of the experiment shown in C. E: Mice bearing exponentially growing LNCaP tumors were randomized (12 mice per group) and received daily i.p. injections of cont-siRNA (black symbols), hAR-siRNA (white symbols) or an oral dose of 50 mg.kg⁻¹ of bicalutamide (grey symbols); tumor volume (cm³, mean±SE). *p<0.05 and **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors. F: On the fourth day of treatment of the experiment shown in E, 6 mice in each group were sacrificed and AR and PSA mRNA levels were quantified in the tumors by qRT-PCR and normalized with cyclophilin A mRNA level. Results are expressed relative to the mean level in control tumors. **p<0.01 comparing hAR-siRNA to cont-siRNA treated tumors.

Figure 2. Silencing of AR in prostate and testes.

A: Upper panels, immunodetection of AR expression in the ventral prostate of mice treated for 3 weeks with hAR-, cont-, or panAR-siRNA as indicated. Lower panels, AR expression in testes from mice sacrificed at the end of the experiments shown in figure 1C, after 2 weeks of treatment (cont- and panAR-siRNA) or treated for 3 weeks with hAR-siRNA. B: AR and GST expression in testes from mice treated for 3 weeks with cont- (black bars) or panAR-siRNA (pAR, white bars). AR and GST levels were quantified by immunoblot, normalized with actin level, (arbitrary units, mean±SE, n = 10). C: Quantification of apoptotic germ cells (insert) in testes collected from mice treated for 2 or 3 weeks (mean number/ 100 seminiferous tubules±SE) with cont- (c) or panAR-siRNA (pAR). **p<0.01 as compared to cont-siRNA treated group.

Figure 3. AR-siRNA-induced inhibition of the growth of castration-resistant prostate tumors.

A: C4-2 or 22RV1 cells grown in 10% FCS were transfected with 10nM of cont- or panAR-siRNA using HiPerfect reagent. Grey box plots: at the indicated time points, AR was detected by indirect immunofluorescence, and its expression level was quantified in at least 200 nuclei from at least 5 different high power fields using the simple PCI software in cont- or panAR-siRNA transfected cells. Values at each time point were compared to the mean fluorescence value of the cont-siRNA transfected cell population, set to 1. Each box plot is composed of 5 horizontal lines displaying the 10th, 25th, 50th, 75th and 90th percentiles. In sister wells, the MTT activity (black bars) in AR-siRNA treated cells was measured and expressed as a fraction of that of cont-siRNA transfected cells. The measured caspases activities (white bars) were expressed using the following formula: (caspases/MTT in AR-siRNA treated cells)/(caspases/MTT in cont-siRNA treated cells). B: Relative VEGF protein content in the medium of LNCaP, C4-2 or 22RV1 cells, 72 h after transfection of cells with control (dark grey bars) or hAR-siRNA (light grey bars) (mean±SE, n = 5 to 7 independent experiments). The medium was not changed after transfection. **p<0.01 as compared to values in cont-siRNA transfected cells set to 1. C: Mice bearing C4-2 vascularized and exponentially growing tumors were randomized and received daily i.p. injections of cont-siRNA (Black symbols), hAR-siRNA (white symbols) or panAR-siRNA (grey symbols); tumor volume (cm³, mean±SE n = 6-12). *p<0.05 and **p<0.01 comparing panAR-siRNA to cont-siRNA treated tumors (stars above the curves) or comparing hAR-siRNA to cont-siRNA treated tumors (stars below the curves). D: Frozen sections of regions at the periphery of tumors from C were immunostained by indirect immunofluorescence with AR, KI67 or Collagen-IV antibodies and observed by epifluorescence. Photographs were taken with a 63x (AR, KI67) or 10x (Coll-IV) objective, using the same exposure parameters for cont- and AR-siRNA treated tumors. A representative tumor from each group is shown. E: Male mice bearing exponentially growing 22RV1 tumors were randomized and received either cont- (Black symbols) or hAR-siRNA (white symbols); tumor volume (cm³, mean±SE, n = 5). *p<0.05 and **p<0.01 comparing panAR-siRNA to cont-siRNA treated tumors. F: Mean microvessels density (number of vessels/arbitrary surface unit) in at least 10 non overlapping high-power fields from tumors treated with cont- (black bars) or AR-siRNA (gray bars) in C4-2 or 22RV1 tumors. Necrotic regions were not included in the study. **p<0.01 as compared to cont-siRNA treated group. G: human VEGF quantification in tumor homogenates from C4-2 or 22RV1 tumors in mice treated with cont-siRNA (black bars) or AR-siRNA (gray bars) (ng/mg of protein, mean±SEM). *p<0.05 as compared to cont-siRNA treated group.

Figure 4. Quantification of siRNA copies in CRCaP tumors and mouse tissues.

A: Dynamic range and sensitivity of the hAR-siRNA antisense strand assay. 10⁴ to 10¹¹ copies of the antisense strand of hAR-siRNA were reverse-transcribed using a stem-loop primer. The product was amplified in the presence of SyberGreen and the cycle threshold (CT) plotted against the log of the number of copies of the input target. B: Detection of the antisense strand of hAR-siRNA in tumors (tum), testis (test), prostate (prost) and liver of mice treated with 3 µg of cont- (c) or hAR-siRNA (AR) injected i.p. in 50 µl of saline. The number of siRNA copies is normalized to the tissue weight (mean±SE n = 3).