Fig 1: Basal CDK1 activity is required for AR-regulated locus transactivation. (A) LNCaP cells in CDS medium were treated for 2 h with CDK1 inhibitor RO-3306 (RO, 10 µM), CDK9 inhibitor (iII, 50 µM) and AR antagonist enzalutamide (ENZ, 10 µM). Total proteins were normalized for blotting. (B and C) LNCaP cells in CDS medium were pre-treated for 2 h with CDK1 inhibitor RO-3306 (0.5, 1, 2.5, 5 and 10 µM), followed by 4 h treatment with DHT (10 nM) as indicated. Total proteins were harvested for blotting (B) and total RNA was harvested for qRT-PCR (C) analyses, respectively. (D and E) LNCaP cells in CDS medium were pre-treated for 2 h with indicated inhibitors (RO-3306: RO, 10 µM; Roscovitine: Ros, 10 µM; CGP74514A: CGP, 10 µM; Olomoucine: Olo, 10 µM; PF: Pfizer compound AG024322, 10 µM; Dinaciclib: Dina, 1 µM; CDK1/2 inhibitor: 1/2, 20 µM; and CDK1/5 inhibitor: 1/5, 100 µM), followed by 4 h treatment with DHT (10 nM) as indicated. Total proteins were harvested for blotting (D) and total RNA was harvested for qRT-PCR analysis (E). (F–H) LNCaP cells in CDS medium were transfected with control siRNA or two independent CDK1 siRNAs (20 nM) for 3 days, followed by 4 h treatment with DHT (10 nM). Total proteins were normalized and assessed by immunblotting (F); cells were harvested for ChIP-qPCR analysis (G); or qRT-PCR analysis was carried out with genes of interest (normalized to the DHT negative samples being set as 1) (H). (I and J) LNCaP cells in CDS medium were pre-treated with CDK1 inhibitor RO-3306 (10 µM) for indicated time points, followed by 4 h treatment without or with DHT (10 nM) as indicated. Total proteins were harvested for blotting (F), or cells were subjected to ChIP for AR and pS81 AR (G).
Fig 2: CDK1-mediated basal pS81 and AR activation are not repressed by direct AR antagonists. (A) Abl and C4-2 cells in CDS medium were treated for 4 h with indicated compounds (CDK1 inhibitor RO-3306 (RO, 10 µM), p300 inhibitor (C646, 10 µM), CDK9 inhibitor (iII, 10 µM); BRD4 antagonist (JQ1, 500 nM), bicalutamide (BIC, 10 µM) and enzalutamide (ENZ, 10 µM) and total proteins were normalized for blotting. (B) 293T cells in CDS medium were co-transfected with AR, alone or with activated CDK1 (CDK-AF) and cyclin B1 vectors (23), followed by overnight treatments with bicalutamide (BIC, 10 µM), enzalutamide (ENZ, 10 µM) and DHT (10 nM) as indicated for blotting. (C) LNCaP cells in CDS medium were treated overnight with nocodazole (Noco, 50 ng/ml), followed by 4 h treatment with RO-3306, bicalutamide or enzalutamide (10 µM each) for blotting. (D) Abl and C4-2 cells in CDS medium were treated for 4 h with CDK1 inhibitor RO-3306 (10 µM) or enzalutamide (ENZ, 10 µM), followed by RNA isolation for qRT-PCR analysis of KLK2 pre-mRNA expression. (E) C4-2 cells in CDS medium were treated with enzalutamide (ENZ, 10 µM) and DHT (10 nM) as indicated for 4 h, followed by ChIP analysis. (F) LNCaP (without or with androgen as indicated), Abl and C4-2 cells in CDS medium were treated for 3 days with a range of doses of enzalutamide (0.4, 1, 2.5 and 10 µM), followed by cell proliferation analysis. The results were normalized to the untreated control that was set as 1. (G) Androgen-sensitive PCa cell line (LNCaP), CRPC cell lines (Abl and C4-2) and AR-negative PCa cell line (PC3) in CDS medium were treated for 3 days with a range of doses of RO-3306 (0.4, 1, 2.5 and 10 µM), followed by cell proliferation analysis. The results were normalized to the untreated control that was set as 1. (H) Proposed model for AR transcriptional activation. AR recruits PP1a to dephosphorylate and mobilize P-TEFb (CDK9/cyclin T) from an inhibitory 7SK complex, which can then phosphorylate RNA polymerase 2 (and associated proteins) for elongation and phosphorylate AR S81. The latter pS81 then enhances p300 binding, histone acetylation, BRD4 binding and further recruitment of P-TEFb to generate a positive feedback loop that sustains transcription of AR-regulated genes. CDK1 generates a basal pool of pSer81 that is needed to initiate this pathway, and increased CDK1-mediated pS81 phosphorylation may drive this pathway at very low androgen levels, or in the presence of AR antagonists, in CRPC.
Fig 3: Increased CDK1 activity drives basal AR activity in CRPC cells. (A) Expression of CDK1 (CDC2) and genes mediating CDK1 activation in CRPC Abl versus LNCaP (GSE11428) and between C4-2 versus LNCaP (GSE63479) under basal (CDS medium) growth conditions. (B) LNCaP, Abl and C4-2 cells in CDS medium were harvested and equal amounts of protein were analyzed by immunoblotting. (C and D) Abl and C4-2 cells in CDS medium were treated with 10 µM RO-3306 for 2 and 4 h, followed by blotting for AR and pS81 (C) and qRT-PCR analyses of KLK2 pre-mRNA (D) expression. (E and F) Abl (E) and C4-2 (F) cells in CDS medium were treated with RO-3306 for 2 h, followed by ChIP analysis of the indicated AR-regulated loci for occupancy by AR, pS81 and Ser5 phosphorylated RNA Pol II (pS5). (G and H) C4-2 cells in CDS medium were transfected with control siRNA, AR siRNA or two independent CDK1 siRNAs (20 nM) for 3 days. Total proteins were normalized and analyzed by immunoblotting (G); or qRT-PCR analysis was carried out for PSA mRNA (value normalized to control siRNA) (H).
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