Fig. 5: Crosstalk between the AR and PKA/SPP1 axis.

A Dispersion plot showcasing a Spearman correlation analysis between AR score and SPP1 mRNA expression in patients with bone metastasis from the SU2C-PCF dataset (n = 83). B Gene expression levels of SPP1 measured by RT-qPCR in PC3 grown alone (PC3) or co-cultured with MC3T3 (PC3/MC3T3) and in C42B grown alone (C42B) or co-cultured with MC3T3 (C42B/MC3T3). Values were normalized using PPIA as a reference gene and relativized to the expression in each control cell line. Statistical significance was assessed via Wilcoxon test. Three independent experiments were performed. Results are shown as the mean ± S.E.M. C SPP1 expression (FPKM) assessed by RNA-seq in MDA PCa 118b and 183 growing intrafemorally (n = 5/group). Statistical significance was assessed using a Wilcoxon test. D-F SPP1, PRKACB and PRKACA gene expression levels assessed by RT-qPCR in PC3 cells transfected with an empty vector (PC3) or a plasmid to overexpress AR (PC3-AR) and treated or not with FK 1 μM and/or DHT 10 nM for 24 h. Values were normalized using PPIA as a reference gene and relativized to controls. Results are shown as mean ± S.E.M. Kruskal–Wallis was used to evaluate statistical significance. G PKA activity was measured by immunofluorescence in PC3 cells transfected with an empty vector (PC3) or a plasmid to overexpress AR (PC3-AR) and treated or not with DHT 10 nM for 24 h. Violin plots showcasing fluorescence intensity. Kruskal–Wallis was used to assess statistical significance. *P < 0.05. H SPP1 mRNA expression levels in C42B cells growing in collagen and fibronectin coated surfaces (C. mix) and treated with enzalutamide (Enz) 30 or 50 µM for 96 h. Values were normalized using PPIA as a reference gene and relativized to controls. Results are shown as mean ± S.E.M. Kruskal–Wallis was used to evaluate statistical significance. I Schematic representation of intrafemoral (i.f.) implantation of MDA PCa 183 PDX or C42B-luc cells in male CB17 SCID mice, followed by surgical castration (or sham-castration as control). Tumor-bearing bones were harvested either 7 weeks or 4 weeks after castration, respectively, for further histological analysis. J Representative photomicrograph images of sections immunostained with OPN derived from MDA PCa 183 PDX or C42B-luc tumors growing i.f. in castrated or non-castrated (control) mice. Magnification 200X. K SPP1 expression in patients with PCa bone or other metastasis from the Westbrook et al. dataset pre (circles) and post (rhombus) enzalutamide treatment. Paired t test was used to assess statistical significance. Paired t test was used to assess statistical significance. L SPP1 expression in patients with PCa bone metastasis with high (cherry) or low (blue) SPP1 levels from the Westbrook et al. dataset pre (circles) and post (rhombus) enzalutamide treatment. M Dot plot showing upstream/master regulators that activate (green) or inhibit (purple) PKA and their P values obtained with IPA in patients with bone metastasis from the Westbrook et al. dataset post vs. pre-enzalutamide, whose SPP1 levels were increased after enzalutamide exposure. The x-axis represents the activation z score. N Cell viability assay in C42B cells pre-treated with forskolin (FK) 1 µM for 0.5 or 24 h and treated or not with enzalutamide 30 or 50 µM for 96 h. Kruskal–Wallis was used to evaluate statistical significance. Three independent experiments were performed. Statistical significance was set at P < 0.05. *P < 0.05, **P < 0.001, ***P < 0.0001, ***P < 0.00001. ns not significant.