Fig. 6. YAP1 inhibitor markedly attenuated growth and cancer stemness in enzalutamide-resistant cells.

A EnzaR#2 cells were treated with 10 μM enzalutamide, darlolutamide, apalutamide or 0.5 μM Verteporfin (VP) for indicated time points and cell proliferation was analyzed by MTS assay (n = 3). Asterisk indicates p < 0.05 using two-way ANOVA test. B EnzaR cells treated with vehicle or VP compound were used to perform tumor sphere assay. Tumor sphere with diameter greater than 50 mm was counted (n = 3). Representative pictures were shown in the left panel. Asterisk indicates p < 0.05 using two-tailed Student’s t-test. C Expression levels of cancer stemness markers were analyzed by using RT-qPCR in enzalutamide-resistant cells treated with different doses of VP compound for 24 h (n = 3). Asterisk indicates p < 0.05 using one-way ANOVA test. D EnzaR (#2) cells with stable luciferase were performed orthotopic injection into the mouse prostate of NOD-SCID mice with castration. Mice were intraperitoneally received 5% DMSO (Con) or VP (25 mg/kg) compound treatment twice a week for 1 month. Tumor growth was analyzed by IVIS spectrum imaging system and result was quantified in the right panel (n = 5 for each group). E LNCaP (WT) or EnzaR cells were pretreated with vehicle or VP (0.5 μM) for 24 h. Cells were individually trypsinized and subcutaneously injected 1 × 10⁴ cells into castrated SCID mice for 4 months. A representative picture of tumor was presented when mice were sacrificed for analysis. Tumor incidence rate in different groups was calculated in the right panel.