Fig 1.
Prostate cancer cells show low Wnt-reporter activity and hormone-deprivation enhances activation of Wnt-reporter in LNCaP cells.
A, -catenin knockdown diminished the Wnt-reporter activation in response to GSK-3 inhibitor. abl cells were infected with eGFP Wnt-reporter (7xTcf-eGFP/SV40-mCherry) and transfected with si-control or si--catenin. Cells were cultured for 2 days and then treated with vehicle, 2 μM or 3 μM GSK-3 inhibitor for 24 h. The relative mRNA levels of eGFP indicating Wnt activity were analyzed by qRT-PCR. B, The levels of nuclear -catenin (green) in LNCaP, abl and 22Rv1 prostate cancer cells were determined by immunofluorescence staining. C, Representative fluorescence images showing the low level of eGFP Wnt reporter activity in LNCaP, abl and 22Rv1 cells: Wnt activity (green), presence of cells (mCherry, red), and nuclear location (DAPI, blue). D and E, Androgen-independent abl and 22Rv1 cells are more prone to activation of Wnt-reporter. Cells were treated with GSK-3 inhibitor for 24 h and subjected to fluorescence imaging (D) or qRT-PCR to measure relative mRNA levels of eGFP (E). In A-E, experiments were performed in the normal growth media of each cell line as described in the Materials and Methods. F and G, Hormone-deprivation enhanced activation of Wnt-reporter in LNCaP cells, which is diminished by androgen treatment. Cells were treated with 9 μM GSK-3 inhibitor in complete (i) or hormone-deprived media with/without 10 nM di-hydrotestosterone (DHT) for 24 h (ii and iii). Cells were then subjected to fluorescence imaging (F), or qRT-PCR to measure relative mRNA levels of eGFP (G). The data presented is representative of three independent experiments and the indicated error is the standard deviation.
Fig 2.
Inhibition of AR activity enhances Wnt/β-catenin target gene expression in response to GSK-3 inhibitor or APC knockdown.
LNCaP cells were treated with 6 μM or 9 μM of GSK-3 inhibitor (A-C); abl cells were treated with and 2 μM or 3 μM of GSK-3 inhibitor (F-H). A and F, Cells were hormone-deprived for 3 days and then treated with vehicle or increasing concentration of GSK-3 inhibitor with/without 10 nM DHT for 24 h. B and G, Cells were treated with vehicle or increasing concentration of GSK-3 inhibitor with/without 10 μM enzalutamide (Enz) for 24 h. C and H, Cells were transfected with si-control or si-AR, cultured for 2 days and then treated with vehicle or increasing concentration of GSK-3 inhibitor for 24 h. D and E, LNCaP cells were transfected with si-control or increasing amounts of si-APC and either hormone-deprived for 2 days and then treated with vehicle or 10 nM DHT for 24 h (D), or cultured in complete media for 2 days and then treated with vehicle or 10 μM enzalutamide for 24 h (E). The relative mRNA levels of indicated genes are analyzed by qRT-PCR. The data presented is representative of three independent experiments and the indicated error is the standard deviation.
Fig 3.
Hormone deprivation increased β-catenin interaction with TCF4 and β-catenin binding to TCF binding sites.
A, Expression of Wnt/-catenin target genes (eGFP reporter and Axin2) and the AR target gene (PSA) was analyzed. LNCaP cells were hormone-deprived for 3 days and then treated with vehicle, 10 nM DHT, 9 μM GSK-3 inhibitor or a combination of both for 24 h. B and C, -catenin or AR binding on target genes was analyzed using chromatin-immunoprecipitation. LNCaP cells were hormone-deprived for 3 days and then treated with vehicle, 100 nM DHT, 9 μM GSK3-i or a combination of both for 4 h. D and E, -catenin interaction with TCF4 or AR was analyzed using co-immunoprecipitation. LNCaP cells were hormone-deprived for 3 days and then treated with vehicle, 100 nM DHT, 9 μM GSK-3 inhibitor or a combination of both for 4 h. The protein lysates were either immunoblotted with indicated antibodies (D) or subjected to co-IP studies (E) Quantification of β-catenin and active β-catenin protein levels are shown below panels in (D). Relative densitometry is normalized to vehicle alone, set to 1. The data presented is representative of three independent experiments and the indicated error is the standard deviation.
Fig 4.
Wnt/β-catenin activation promotes androgen-independent growth of LNCaP cells and inhibition of both AR and Wnt/β-catenin pathways shows increased growth inhibition of LNCaP cells.
A-F, GSK-3 inhibitor treatment or APC knockdown promotes growth of LNCaP cells in hormone-deprived or enzalutamide (Enz) treated media. A and B, Cells were hormone-deprived for 3 days and then treated with vehicle, 10 nM DHT or 6 μM GSK3-i (A), or 10 nM DHT with/without 10 μM Enz or 6 μM GSK3-i (B) every two days. C and D, Cells were treated as described in (A) or (B) for 24 h and then the relative mRNA levels of UBE2C were analyzed by qRT-PCR. E and F, Cells were transfected with si-control or si-APC, hormone-deprived for 2 days and then treated with vehicle or 10 nM DHT (E), or 10 nM DHT with/without 10 μM Enz (F) every two days. G and H, Treatment of cells with Wnt/β-catenin inhibitor (C3) diminishes the growth-promoting effect of GSK3-i. LNCaP cells were hormone-deprived for 3 days and then treated with vehicle or 6 μM GSK3-i (G), or 10 nM DHT plus 10 μM Enz (H) with/without increasing concentrations of C3 every two days. I, Co-treatment of Enz and C3 shows increased growth inhibition of LNCaP cells. Cells were hormone-deprived for 3 days and then treated with 10 nM DHT with/without 5 μM or 10 μM C3, 0.1 μM Enz or a combination of both compounds every two days. Cell viability is represented by relative fluorescence units normalized to time zero (prior to treatments). The data presented is representative of three independent experiments and the indicated error is the standard deviation.
Fig 5.
Inhibition of both AR and Wnt/β-catenin pathways shows increased growth inhibition of abl cells.
A and B, Wnt/β-catenin activation shows no effect on growth of abl cells. Cells were treated with vehicle, 10 nM DHT or 6 μM GSK-3 inhibitor (GSK3-i) (A), or vehicle or 10 μM enzalutamide (Enz) with/without 6 μM GSK3-i (B) every two days. C, Inhibition of the Wnt/β-catenin pathway through β-catenin knockdown sensitized abl cells to Enz. Cells were stably infected with lentiviral vectors encoding control shRNA (sh-control) or -catenin shRNA (sh--cat-1 and 2) and cultured with treatment of vehicle, 0.1 μM or 1 μM Enz every two days. D, Co-treatment of Enz and Wnt/β-catenin inhibitor (C3) shows increased growth inhibition of abl cells. Cells were treated with vehicle, 5 μM or 10 μM C3, 10 μM Enz or a combination of both compounds every two days. Cell viability is represented by relative fluorescence units normalized to time zero (prior to treatments). The data presented is representative of three independent experiments and the indicated error is the standard deviation.