Fig 2: Optimal conditions for differentiation of PC cells to NEPC cells.PC cells were cultured in control medium (Crtl), phenol-red-free medium supplemented with 10% charcoal-stripped FBS (CS-FBS), or control medium supplemented with epinephrine (EPI), forskolin (FSK), IL-6, or both IL-6 and EPI. a Cells were imaged on day 3 following culturing in the respective media. Cells were cultured for 7 days in either control or NE-inducing medium, and expression of CgA and NSE were quantified by qPCR in b LNCaP, c C4–2, and d DU145 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 e Cells were cultured for 7 days in either control medium or phenol-red-free medium supplemented with 10% CS-FBS medium. NSE and CgA were analyzed by western blot. f MTT assays to detect viability of LNCaP, C4–2 and DU145 cells and their corresponding NEPC cells in the presence of 100 nM paclitaxel at 0, 12, 24, 36, and 48 h. *P < 0.05. Error bars represent the standard deviation of biological triplicates

Fig 3: H19 induces epigenetic changes including modifying histone methylation by binding to the PRC2 complex.A Nuclear localization of H19 in NCI-H660. y-Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, BRN2, and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16DCRPC and AdPC cell line LNCaP after transient overexpression of H19. The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16DCRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19FL (full-length H19) or H19DEL (5' deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD (A, C), or mean ± SEM (E); n = 3 (A, C, E) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.

Fig 4: Diagnostic and predictive ability of H19.A Assessment of sensitivity and specificity of H19 in NEPC for use as a diagnostic along with other recently identified NEPC oncogenes (BRN2, SRRM4, and PEG10). Arrows denote patients that are negative for CHGA (black) or CHGA, SYP, and NSE (red) yet positive for H19. B, C, Kaplan–Meir estimates for the impact of H19 on prostate cancer clinical outcomes. B Survival analysis for H19 in ADT untreated patients and C ADT treated patients for biochemical recurrence (BCR—left panels) and metastasis (MET—right panels). Samples were split by H19 tertile expression to test if either of these subgroups of patients were more likely to develop BCR and MET in the context of treatment. P values were calculated using a Kaplan–Meier estimator statistical test to determine significance for the observed stratification in probabilities across the three subgroups in each panel. D, Illustration of H19 transcription and mechanism of action during PCa, NEtD, and tNEPC. During early stage disease, adenocarcinoma cancer cells are driven by an active AR bound to androgens that prevent the transcription of H19. As the disease progresses (gray triangle), ADTs suppress AR activity, SOX2 increases, and results in the persistent transcription, expression, and activation of H19. Once active, H19 operates via dual epigenetic mechanisms; (on the left) binds PRC2 complex members, aiding in altering methylation on H3K4Me3/H3K27Me3 histone marks of PRC2 target genes and (on the right) genome-wide alteration of methylation at CpG sites on DNA. Collectively, these epigenetic changes result in the activation of NE gene expression and deactivation of AR signaling gene expression.

Fig 5: EZH2 is required for NEPC cells.a Expression profiles of PRC subunits in PAC and NEPC samples. P-value adjusted by Bonferroni method. **P < 0.01, ****P < 0.0001. (All values below 0 were close to 0 and the green color was therefore not distinguishable on the right bar). b qPCR analysis of EZH2 and PHF19 expression in our tumor specimen cohort including 40 PAC and eight NEPC samples. ****P < 0.0001. c qPCR analysis of expression of four PRC genes in control PC and NEPC cells. **P < 0.01, ****P < 0.0001. d Western blotting of EZH1 and EZH2 in control PC and NEPC cells. e ChIP enrichment analysis of EZH2-binding site on miR-708 in control PC cells and NEPC cells. f Effect of EZH2 knockdown and EZH2 inhibition by GSK-126 on NEPC markers expression. EZH2-knockdown PC cells were treated with NEPC-inducing medium for 6 days, or control cells were treated with NEPC-inducing medium with GSK126 (2.5 µM) for 6 days. NEPC phenotype was evaluated by qPCR of NEPC markers CgA and SCG3. g Effects of EZH2 inhibitor GSK126, docetaxel (DTX), PI3K/AKT pathway inhibitor LY294002 alone, combination of GSK126 with DTX or LY294002, or DTX + LY294002 on cell viability of NEPC cells derived from C4–2. NEPC cells were treated with vehicle, GSK126 (5 µM), DTX (10 nM), LY294002 (LY, 5 µM), DTX + LY294002, DTX + GSK126, or DTX + GSK126 + LY for 48 h, and the percentages of viable cells were counted and calculated. *P < 0.05, **P < 0.01. h miR-708 expression in NEPC cells derived from C4–2 treated with the indicated drugs for 24 h. **P < 0.01, ****P < 0.0001. i Effects of anti-miR-708 on NEPC markers expression when EZH2 was inhibited. C4–2 cells were transfected with anti-miR-708 and then treated with NEPC-inducing medium for 6 days with or without GSK126. **P < 0.01. Error bars represent the standard deviation of biological triplicates