SphK1 and Androgen Signaling Figure 2. Effect of castration on tumor growth and SphK1 activity of established GFP-expressing human LNCaP and PC-3 cells in SCID mice. Nine and three weeks after SOI of LNCaP/GFP and PC-3/GFP cells respectively, SCID mice were randomized into two groups. These animals were then subjected to castration or sham treatment. Representative fluorescent primary LNCaP (A) and PC-3 (C) tumors from sham- and castration-treated animals at the time of autopsy (7 days post-treatment). Tumor mass of excised primary LNCaP (B, left panel) and PC-3 (D, left panel) GFP-labeled tumor. Columns, means from 8 animals; bars, SE. SphK1 activity was measured in tissue extracts obtained from sham-, and castration-treated LNCaP (B, right panel) and PC-3 (D, right panel) tumor bearing animals. Columns, means from 8 animals; bars, SE. The two-tailed P values between the means are as follow: ***, P,0.001; or ns, not significant. doi:10.1371/journal.pone.0008048.g002 Consistent with previous observations [41,44], immunoblots showed that CgA expression was very low in both parental LNCaP (Fig. 6A, lower panel) and C4-2B (Figure S1) cells, while its expression level was greatly enhanced over time in hormone- deficient medium. Furthermore, compared to non hormone- deprived LNCaP cells (D0), a 3-fold increase in NSE secretion to the medium was found after 42 days under CSS conditions indicative of a NE-like phenotype (Fig. 6A, right panel). Based on our previous observations that SphK1 activity upon androgen deprivation was only transiently inhibited with a significant rebound within 4 days of incubation in CSS medium (Fig. 1D), we then analyzed the SphK1 activity up to 42 days during the NE transdifferentiation process. Remarkably, SphK1 activity (Fig. 6B) increased progressively up to a 4-fold increase after 42 days of androgen depletion. This was further illustrated by a marked increase in S1P content (Fig. 6B, inset). In parallel, SphK1 protein expression was significantly increased (Fig. 6B, right panel). The activation of SphK1 was correlated with stimulation of Akt (Fig. 6B, right panel), a pro-survival signaling pathway reported to be up-regulated during NE transdifferentiation of LNCaP induced by androgen depletion [6,7,23,45]. Comparable findings were observed in the C4-2B cell model (Figure S1). In line with previous studies [6], pharmacological inhibition of the PI3K/Akt pathway could impede NE transdifferentiation of both LNCaP (Fig. 6C) and C4-2B (Figure S1) maintained for 10 days under CSS conditions. Of note, the blockade of CgA accumulation induced by the PI3K inhibitors WT and LY294002 was markedly associated with a decrease in both Akt phosphor- ylation and SphK1 activity (Fig. 6C, right panel and Figure S1). To investigate the functional role of SphK1 in NE transdiffer- entiation, we examined the effect of the pharmacological inhibitor SKI-2 on LNCaP and C4-2B cells. In presence of SKI-2 (5 mM), PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e8048