Fig 2: TNIK inhibitor inhibited proliferation and invasion of CRPC cell(A) C4-2 and PC3 cells were treated with NCB-0846 24 h. Whole-cell extracts were analyzed by western blotting with the indicated antibodies.(B) C4-2 and PC3 Cells were seeded into 96-well plates and treated with NCB-0846 (10 μm); MTT detected cell viability. Data are shown as mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.005.(C) C4-2 and PC3 cells were treated with NCB-0846 (10 μm); transwell detected cell invasion ability. Scale bars: 100 μm. Data are shown as mean ± SD. ∗∗∗p < 0.005.

Fig 3: Targeting TNIK suppresses CRPC tumor progression in vivo(A) C4-2 cells were implanted subcutaneously in male BALB/c mice. When tumors became palpable, mice administered daily by oral gavage either with vehicle (10% DMSO in PBS) or NCB0846 (80 mg/kg of body weight) for 10 days (n = 4 mice for each treatment). Tumor volumes were measured with calipers.(B) Tumor size of xenografts of the above represented the growth of tumor over 10 days (n = 4) in athymic nude mice (p < 0.001). Data are shown as mean ± SD.(C) Tumor weight of the control mice tumors and NCB-0846-treated mice tumors (p < 0.001). Data are shown as mean ± SD.(D) Body weight of nude mice after implantation of control or C4-2 xenografts and treatment with vehicle or NCB-0846 for 4 weeks.(E) Quantitation of Ki-67, TNIK, p-EGFR, β-catenin, vimentin, E-cadherin, BMP6, and BMP7 expressions in C4-2 xenograft tumors from each group; specimens were got at 10 days posttreatment. Scale bars: 500 μm. The IHC was scored according to number of cells expressing the indicated proteins, and statistical analysis was performed (non-parametric Kruskal-Wallis test) in order to determine significance. Data are shown as mean ± SD. ∗∗∗p < 0.005.

Fig 4: TNIK was upregulated in castration-resistant prostate cancer(A) Heatmap of the genome-wide transcript profile between (CR-LNCaP) castration tumors and (HS-LNCaP) uncastration tumors.(B) GO analysis of transcript profile.(C) qPCR detects mRNA expression of TNIK in (CR-LNCaP) castration tumors and (HS-LNCaP) uncastration tumors (p < 0.05). Data are represented as mean ± SD.(D) Clinical specimens of benign prostate hyperplasia (BPH), hormone native, and CRPC were analyzed by IHC for TNIK expression. Scale bars: 200 μm. H-score of TNIK immunoreactivity in HSPC, CRPC, and their paracancerous tissues. Data were presented as median (interquartile range). Data are represented as mean ± SD. The hash symbol indicates significantly different from Para (HSPC) group (p < 0.05); an asterisk indicates significantly different from Para (CRPC) group (p < 0.05).

Fig 5: The essential role of the AR and H3K27me3 complex in the regulation of TNIK expression(A) LNCaP cells were cultured in steroid-depleted medium for 24 h and subsequently treated with DHT (10 nM) for the indicated time. Protein levels were analyzed by western blot.(B) The levels of TNIK protein were measured by western blot in LNCaP and C4-2 cells upon AR knockdown.(C) qPCR analysis mRNA expression of TNIK after LNCaP cells were treated with MDV3100 for 24 h. Data are shown as mean ± SD. ∗∗∗p < 0.005.(D) The levels of TNIK protein were measured in LNCaP cells treated for 24 h with the AR antagonist MDV3100 (enzalutamide, 100 nM).(E) Coimmunofluorescence (co-IF) analysis of TNIK and AR proteins in LNCaP treated with MDV3100. Scale bars: 50 μm.(F) Immunoprecipitation of AR or H3K27me3 in LNCaP and C4-2 cells followed by immunoblot analysis of H3K27me3 or AR. IgG represents a control antibody used for IPs.(G) ChIP–PCR analysis of H3K27me3 and AR binding to the TNIK gene promoter in LNCaP cells treated with MDV3100. ChIP–PCR primer in Table S3. Data are shown as mean ± SD. ∗∗∗p < 0.005.(H and I) LNCaP and C4-2 cells were treated with DZNep (0, 5, 10 μm) and GSKJ1 (0, 5, 10 μm) for 8 h, WB analysis of TNIK protein expression.(J) Western blot showed that treatment of the indicated LNCaP or C4-2 cells with GSK-J1 could reverse the effects of AR knockdown on TNIK.