Figure 7. Concerted Activation of AKT and GATA2/AR signaling is essential for mediating MAPK4 tumor-promoting activity in PCa.
(A) Western blots on LAPC4 cells and VCaP cells with knockdown of MAPK4 (G2, G4) or control (NT). (B) Western blots, (C) qPCR, (D) Proliferation assays, and (E) Soft-agar assays (original magnification: × 50) on the engineered LNCaP cells with 0.5 μg/mL Dox-induced overexpression of MAPK4 (iWT), MAPK4S186A (iS186A), MAPK4D254A (iD254A), or control (iCtrl). Data represent mean ± SEM. Adjusted P values determined by 1-way ANOVA followed by Dunnett’s multiple comparisons. ***P ≤ 0.001. ****P ≤ 0.0001. (F) Proliferation assays and Western blots on the LNCaP cells with 0.5 μg/mL Dox-induced overexpression of MAPK4 (iWT), MAPK4D254A (iD254A), or control (iCtrl), treated with 1 μM of AKT inhibitor MK2206 or DMSO control. (G) Proliferation assays and Western blots on the LNCaP cells with 0.5 μg/mL Dox-induced expression of MAPK4, MAPK4D254A, or control, also infected with lentivirus expressing AKT1-DD mutant or control. (H) Proliferation assays on the LNCaP cells with 0.5 μg/mL Dox-induced overexpression of MAPK4, MAPK4D254A, GATA2, or control, also overexpressing AKT1-DD or control. The growth of these cells in 10% FBS, 5% CSS, and 5% CSS plus 1 nM R1881 were compared. Also shown are Western blots on these cells cultured in 10% FBS. (I) Proliferation assays and Western blots on the control (NT) or MAPK4-knockdown (G2) VCaP cells with 0.5 μg/mL Dox-induced expression of GATA2 (iGATA2), AKT1-DD (iAKT1DD), or control (iCtrl). Data are representative of at least 3 independent experiments.