SphK1 and Androgen Signaling Figure 1. Effect of short-term androgen deprivation on cell growth and SphK1 activity. Overnight serum deprived LNCaP (A, D) and PC-3 (C, D) cells were incubated in presence of 5% FBS, 5% CSS or without serum (SFM) for the indicated times. Cell proliferation in LNCaP (A, left panel) and PC-3 cells (C) was determined by MTT assay and expressed as percent of control at the beginning of the experiment (Day 0). Cell number (A, middle panel) and DNA synthesis (A, right panel) were measured as described in Materials and Methods. Columns, mean of at least twenty-four independent experiments for MTT assay and six experiments for cell counting and DNA synthesis; bars, SD. The P values between the means are as follow: ***, P,0.001. Secreted PSA level was measured in culture media from LNCaP cells (B). Columns, mean of at least six independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001. D, SphK1 activity was determined in both LNCaP and PC-3 cells and expressed as percent of FBS-treated cells. Columns, mean of at least twelve and six independent experiments for LNCaP and PC-3 cells respectively; bars, SD. The P values between the means are as follow: ***, P,0.001; **, P,0.01; *, P,0.05; ns, non significant. doi:10.1371/journal.pone.0008048.g001 Dihydrotestosterone Rapidly and Transiently Stimulates SphK1 Activity in an Androgen Receptor/PI3 Kinase-Dependent Manner As a down-regulation of SphK1 activity was correlated with the removal of androgens in LNCaP cells (Fig. 1D), it was of interest to establish whether this could be reversed by the addition of androgens. Under CSS conditions, the addition of dihydrotestosterone (DHT) could trigger an early and transient stimulation of SphK1 (as early as 15 min) after which SphK1 activity returned to basal levels (Fig. 4A). The rapid stimulation of SphK1 was dependent on activation of the PI3K/Akt signaling, which has been shown to mediate the rapid effects of androgens [39,40]. Indeed, while wortmannin (WT) inhibited both the activation of Akt (Fig. 4B, top) and SphK1 (Fig. 4B, bottom) induced by DHT, the SphK1 inhibitor SKI-2 did not impact Akt phosphorylation (Fig. 4B, top). The stimulation of the PI3K/Akt/SphK1 pathway was critical for transmitting the proliferative effects of DHT under CSS conditions. In fact, similar to the PI3K inhibitors WT and LY294002 (Fig. 4C), the SphK1 inhibitors SKI-2 and F-12509a could impede DHT-induced cell proliferation, DNA synthesis as well as PSA secretion (Fig. 4D). The effect of androgen receptor antagonist was next examined with bicalutamide. As anticipated, LNCaP cell proliferation response at 10 nM DHT was totally blunted by 10 mM bicalutamide (Fig. 5A). The DHT-triggered stimulation of SphK1 activity was also completely inhibited in presence of androgen receptor antagonist (Fig. 5B). SphK1 Is Involved in Androgen Depletion-Induced Neuroendocrine Transdifferentiation of LNCaP and C4-2B Cells We next investigated the potential involvement of SphK1 in the transition to androgen refractory state after chronic androgen withdrawal. To this end, LNCaP and C4-2B cells, which have been previously reported to acquire a neuroendocrine (NE) phenotype [20,21,23,41,42,43] were maintained under CSS conditions for up to 42 days. LNCaP cells exposed to a hormone-deficient medium underwent neuroendocrine (NE) morphological changes (apparent after approximatively 7–10 days) as indicated by soma compaction and development of long and branched neuritic extensions (Fig. 6A), whereas control LNCaP parental cells retained a fusiform epithelial morphology (Fig. 6A). In addition to morphological characteristics, transdiffer- entiated NE-like prostate cancer cells are defined by the expression of a number of neurosecretory products including chromogranin A (CgA) and neuron-specific enolase (NSE). CgA is considered to be the most reliable indicator of prostatic NE differentiation. PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e8048