Fig. 3

ADT activates EZH2 through PKA/CREB signaling. a Western blotting for LNCaP-Dox-shCREB cells treated with 10 μM MDV3100 without or with 1 μg ml⁻¹ Dox for 72 h. b Elevated H3K27me3 levels in PC3 cells overexpressing wild-type (WT) or constitutively active mutant (Y134F) of CREB cDNA. c LNCaP cells were treated with PKA/CREB signaling activators, i.e., 10 μM forskolin (FSK, adenylyl cyclase activator) with 0.5 mM IBMX (phosphodiesterase inhibitor) for 24 h (left). Similar experiments for three other cell lines (PC3, NE1.3, and RWPE) treated with these PKA/CREB activators are shown in Supplementary Fig. 1a. Conversely, NEPC cells 144-13 were treated with PKA inhibitor PKI, beta-adrenergic antagonist ICI or propranolol (PRO) (all 10 μM, 48 h) (right). Similar experiment on another NEPC line NE1.3 was shown in Supplementary Fig. 1b. d, e RT-qPCR analyses of EZH2 target genes DAB2IP and ADRB2 in LNCaP cells treated with 10 μM PKA signaling activator forskolin or ISO (d), and in NE1.3 cells treated with PKA signaling inhibitor ICI, or PRO (e) (all 10 μM, 48 h). f Western blotting of LNCaP cells treated as indicated for p-CREB, H3K27me3, and NE marker ENO2. 10 μM MDV3100 with or without PRO + ICI (CREB signaling inhibitor, 10 μM each) or 5 μM EPZ6438 (EZH2 inhibitor) for 72 h. 5 nM of DHT was added in one of the MDV3100 treated cells 24 h before sample collection. Similar results were observed in another AR-positive cell line 22Rv1 (Supplementary Fig. 2a). g A tissue microarray with 78 cases of human prostate cancer and normal samples was IHC stained with antibodies against pS133-CREB and H3K27me3. IHC pictures for two representative cases are shown (scale bar = 50 μm). The summary of IHC data is in Supplementary Table 1