Fig. 5: NKX2-1 is highly expressed in NEPC tumors and accelerates NET of PCa.
a, Volcano plots showing human TFs differentially expressed in CRPC versus NEPC. X axis represents log2 of FC, while the y axis shows −log10 of q values. Each dot represents a TF, red dots—log2(FC) ≥ 1 and adjusted P < 0.05. Adjusted P values by Wald test with Benjamini–Hochberg correction. b, NKX2-1 and FOXA2 gene expression in human PCa samples with distinct expression of AR and NE genes. P values by two-sided Wilcoxon test. c, IHC of NKX2-1 and FOXA2 proteins in clinical CRPC TMAs. Representative IHC images (n = 3) are shown in ×10 with the insets shown in ×40 (left). Scale bar = 100 µm. IHC staining intensities in AR+NE−, AR−NE−, AR+NE+ and AR−NE+ samples are quantified on the right. d, Concomitant NKX2-1 OE accelerated FOXA2-driven NET. LNCaP cells were infected with FOXA2, NKX2-1 or both. Cells were collected at the indicated time points and analyzed by WB (n = 3). e, Heatmap showing FOXA2 ChIP–seq in LNCaP cells with OE of FOXA2 alone, or FOXA2 and NKX2-1 at D14 or D28. Peaks were centered around (±2 kb) the six ATAC–seq clusters and sorted by FOXA2 ChIP–seq intensity. Scale bar—enrichment intensity. f, WB (left, n = 3) and RT–PCR (right) showing NKX2-1 KD decreases the expression of NE lineage markers in NCI-H660. NCI-H660 cells were transfected with control or NKX2-1 siRNAs and collected on day 5 after transfection for WB and RT–PCR analyses. RT–PCR data were normalized to GAPDH. Shown are the mean ± s.e.m. of technical replicates from one of three (n = 3) independent experiments. P values by two-sided t test. g, WB (left, n = 3) and RT–PCR (right) showing NKX2-1 KD abolishes RB1/TP53-KD-induced NET. LNCaP cells were co-infected with sgRB1 and sgTP53 virus along with either sgNC or sgNKX2-1 virus. The infected cells were collected at 4 weeks after infection for analyses of NE and stem cell lineage markers. Data of RT–PCR are shown as in f. h, RT–PCR (left) and WB (right, n = 3) showing NKX2-1 OE promotes NET in ENZ-resistant AR+/PSA− cell line 42D. The infected cells were collected at 4 weeks after infection for analyses of NE lineage markers. Data of RT–PCR are shown as in f.
