Inhibition of glycogen synthase kinase-3β counteracts ligand-independent activity of the androgen receptor in castration resistant prostate cancer

Abstract

In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.

Figure 1. Intracellular levels of AR, GSK-3β and PSA in different PC cell lines.

(A) Intracellular AR and GSK-3β-protein levels in PC cell lines grown in the absence of androgens. The AR-positive LNCaP, LNCaP-SSR and C4-2 cells as well as the AR-negative PC3 cells were grown for 48 hours under androgen-deprived conditions. Subsequently, cells were lysed and cell lysates were analyzed by Western blotting for AR and GSK-3β. β-actin bands served as loading control. (B) Intracellular PSA levels in CPRC cells grown under androgen-deprived conditions. AR-positive LNCaP, LNCaP-SSR and C4-2 cells were grown and treated as described. Relative PSA levels were determined in the corresponding cell lysates by Western blotting as described in Material and Methods. β-actin bands served as loading control.

Figure 2. SB216763 inhibits phosphorylation of GSK-3β at tyrosine 216.

LNCaP cells and C4-2 cells were seeded into T25 flasks and allowed to adhere overnight. After that, medium was replaced by steroid-free medium and cells were grown for another 24 hours. Subsequently, SB216763 (final concentration 5 µM) was added 30 min prior to DHT (10 nM) treatment. After 4 hours, cell lysates were analyzed for GSK-3β^Y216 and GSK-3β^S9 phosphorylation as described in Material and Methods.

Figure 3. Inhibition of GSK-3β by SB216763 enhances nuclear export of the AR.

Cells were grown under steroid-free conditions for 24 hours. Cells were then treated with 10 nM DHT and incubated for another 30 min. Subsequently, SB216763 was added (end concentration 5 µM) and cells were allowed to grow for another 210 min. Following this treatment, nuclear extracts were prepared and analyzed for AR and lamin A as described in Material and Methods. AR and lamin A levels were quantified by densitometry. AR levels were expressed in fold-change AR/Lamin of cells grown in absence of DHT and SB216763, which was set at 1.

Figure 4. Effect of SB216763 on the localization of unliganded AR-Eos fusion protein in LNCaP and C4-2 cells.

LNCaP cells and C4-2 cells were transfected with pAR-t1EosFP and grown for 24 hours in the absence of androgens. Thereafter, cells were treated with/without DHT (final concentration 10 nM). After 4 hours nuclear or cytoplasmic localisation of AR-EosFP was monitored by fluorescence microscopy.

Figure 5. Treatment with leptomycin B reverses nuclear export of the AR induced by SB216763 in C4-2 cells.

C4-2 cells grown in the absence of androgens were incubated with/without the CRM1 inhibitor leptomycin B (LMB) 30 min prior to SB216763 treatment. After 4 hours, nuclear extracts were prepared and analyzed for AR and lamin A as described in Material and Methods.

Figure 6. Long term inhibition of GSK-3β in C4-2 cells.

(A) Silencing of GSK-3β in C4-2 cells. C4-2 cells were transiently transfected with pKD-GSK-3β-v1 or pKD-NegCon-v1. 48 hours after transfection, 10 nM DHT was added where indicated. After another 24 hours, nuclear extracts were prepared as described in Material and Methods. (B) Long term inhibition of GSK-3β using SB216763. AR-positive C4-2 cells were treated with increasing amounts of SB216763 for 48 hours in the absence of androgens. Cells were lysed and cell lysates were analyzed by Western blotting for AR. β-actin bands served as loading control.

Figure 7. Inhibition of GSK-3β by SB216763 inhibits the proliferation in CRPC cells.

AR-positive LNCaP, LNCaP-SSR and C4-2 cells as well as the AR-negative PC3 cells, were treated with increasing amounts of SB216763 for 48 hours (A) or with/without 1 µM SB216763 for 0, 24 and 96 hours (B). Proliferation was measured using an MTT assay as described in Material and Methods. Results shown under (A) are expressed as % of untreated controls ± standard deviation. Results shown under (B) are expressed in percent of SB216763-treated/untreated control which was set at 100% for the time point zero ± standard deviation.

Figure 8. Inhibition of GSK-3β diminishes nuclear localization and function of the AR in vivo.

The CAM assay was performed with C4-2 cells. Subsequently, nuclear localization of the AR and intracellular PSA distribution in untreated and SB216763-treated tumor-nodules (magnification 400x) was performed as described in Material and Methods. AR-positive cells are marked with stars.