Inhibition of Glycogen Synthase Kinase-3β Counteracts Ligand-Independent Activity of the Androgen Receptor in Castration Resistant Prostate Cancer

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

IκB Kinases Modulate the Activity of the Androgen Receptor in Prostate Carcinoma Cell Lines12

Enhanced nuclear localization of nuclear factor κB (NF-κB) in prostate cancer (PCa) samples and constitutive NF-κB signaling in a class of PCa cell lines with low androgen receptor (AR) expression (PC3 and DU-145) imply an important role of the IκB kinase (IKK)/NF-κB system in PCa. However, most PCa and PCa cell lines depend on the activity of the AR, and the role of NF-κB in these AR-expressing PCa remains unclear. Here, we demonstrate that inhibition of NF-κB signaling by the IKK inhibitor BMS345541 reduced proliferation and increased apoptosis in AR-expressing PCa cell lines. Furthermore, AR activity and target gene expression were distinctively reduced, whereas AR protein levels remained unaltered on BMS345541 treatment. Similar effects were observed particularly after small interfering RNA (siRNA)-mediated knockdown of IKK1, but not by siRNA-mediated suppression of IKK2. Moreover, IKK1 overexpression augmented 5α-dihydrotestosterone-induced nuclear AR translocation, whereas nuclear AR was reduced by IKK1 knockdown or BMS345541. However, because IKK1 also enhances the activity of a chronically nuclear AR mutant, modulation of the subcellular distribution seems not to be the only mechanism by which IKK1 enhances AR activity. Finally, reduced in vivo AR phosphorylation after BMS345541 treatment and in vitro AR phosphorylation by IKK1 or IKK2 imply that AR constitutes a novel IKK target. Taken together, our data identify IKK1 as a potentially target structure for future therapeutic intervention in PCa

Inhibition of Glycogen Synthase Kinase-3 in Androgen-Responsive Prostate Cancer Cell Lines: Are GSK Inhibitors Therapeutically Useful?

The glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase widely expressed in mammalian tissues. Ini-tially identified by its ability to modulate glycogen synthesis, GSK-3 turned out to be a multifunctional enzyme, able to phosphorylate many proteins, including members of the steroid receptor superfamily. Although GSK-3 was shown to phosphorylate the androgen receptor (AR), its effects on AR transcriptional activity remain controversial. Analysis of short hairpin RNA (shRNA)–mediated downmodulation of GSK-3 proteins in prostate cancer cells showed a reduction in AR transcriptional activity and AR protein levels. Pharmacological GSK-3 inhibitors such as the maleimide SB216763 or the aminopyrazole GSK inhibitor XIII inhibited AR-dependent reporter gene activity and AR expression in vitro. Analysis of androgen-induced nuclear translocation of the AR was performed in PC3 cells transfected with pAR-t1EosFP coding for EosAR, a green fluorescent AR fusion protein. When grown in pres-ence of androgens, EosAR was predominantly nuclear. Incubation with SB216763 before and after androgen treat-ment almost completely reduced nuclear EosAR. In contrast, the thiazole-containing urea compound AR-A014418 increased rather than decreased AR–expression/function. Although not all GSK inhibitors affected AR–stability/ function, our observations suggest a potential new therapeutic application for some of these compounds in pros-tate cancer

A new and reliable culture system for superficial low-grade urothelial carcinoma of the bladder

Several bladder cancer culture systems have been developed in recent years. However, reports about successful primary cultures of superficial urothelial carcinomas (UC) are sparse. Based on the specific growth requirements of UC described previously, we developed a new and reliable culture system for superficial low-grade UC. Between November 2002 and April 2006, 64 primary cultures of bladder cancer specimens were performed. After incubating the specimens overnight in 0.1% ethylenediaminetetraacetic acid solution, tumour cells could easily be separated from the submucosal tissue. Subsequently, cells were seeded in a low-calcium culture medium supplemented with 1% serum, growth factors, non-essential amino acids and glycine. The malignant origin of the cultured cells was demonstrated by spectral karyotyping. Overall culture success rate leading to a homogenous tumour cell population without fibroblast contamination was 63%. Culture success could be remarkably enhanced by the addition of glycine to the culture medium. Interestingly, 86.4% of pTa tumours were cultured successfully compared to only 50% of the pT1 and 38% of advanced stage tumours, respectively. G1 and G2 tumours grew significantly better than G3 tumours (86, 73 and 41%, respectively). Up to three passages of low-grade UC primary cultures were possible. We describe a new and reliable culture system, which is highly successful for primary culture and passage of low-grade UC of the bladder. Therefore, this culture system can widely be used for functional experiments on early stage bladder cance