SphK1 and Androgen Signaling Figure 3. SphK1 overexpression renders LNCaP cells more resistant to short-term androgen deprivation. SphK1 expression in LNCaP cells was analyzed by Western blotting using anti-FLAG antibody (A, left panel). Resting SphK1 activity was measured in LNCaP/neo and LNCaP/ SphK1 cells (A, right panel). B, overnight serum deprived LNCaP cells were incubated in presence of 5% FBS or 5% CSS for 6 days. Cell proliferation was then determined and expressed as percent of 5% FBS-treated cells. Columns, mean of at least twelve independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001. C, secreted PSA level was measured in culture media from LNCaP/Neo and LNCaP/SphK1 cells after 24 h of incubation in 5% CSS medium. Columns, mean of at least six independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001. doi:10.1371/journal.pone.0008048.g003 the distinct NE-like morphology observed was essentially abolished with LNCaP exhibiting a rounded morphology with short rarely branched cellular processes (not shown). Moreover, both secretion of NSE (Fig. 6D, left panel) and CgA accumulation (Fig. 6D, right panel and Figure S1) were markedly diminished while Akt phosphorylation was unchanged hence suggesting that SphK1 inhibition can to a certain extent block the NE transdifferentiation. Physiological or pharmacological agents that increase intracel- lular levels of cyclic AMP (cAMP) can induce the development of a NE morphology in LNCaP or C4-2 cells in presence of steroids [41,44,46]. Of note, cAMP increase has been previously reported to be associated with activation of SphK1 in osteoblasts [47]. Hence, we examined the role of cAMP as a potential upstream regulator of SphK1 during NED. LNCaP and C4-2B cells were stimulated with the adenylate cyclase activator forskolin (Fsk) and the phosphodiesterase inhibitor IBMX, or with epinephrine (Epi), a b-adrenergic receptor agonist. The treatment with Fsk/IBMX or Epi strongly stimulated SphK1 activity (Fig. 7A), and correlated with increased cAMP content (Fig. 7B) and acquisition of NE morphology (data not shown) characterized biochemically by CgA accumulation (Fig. 7C). Interestingly, the pharmacoligical inhibi- tion of SphK1 did not have any effect on cAMP levels (Fig. 7B) whereas it did markedly inhibit NED (Fig. 7C). Remarkably, under androgen deprivation conditions, a similar increase in cAMP intracellular levels was observed, which could not be blocked by pharmacological inhibition of SphK1 (Fig. 7D). These results evoke a role for cAMP for the stimulation of SphK1 activity during NED. In order to determine whether the in vitro findings of the potential involvement of SphK1 in NE differentiation had any relevance to human disease, sections from a representative patient who underwent palliative transurethral resection for local recurrence under complete androgen blockade were immuno- stained for CgA and SphK1 expression (Fig. 8). SphK1 staining was restricted to cytoplasm with some cells being more intensely stained than the others (Fig. 8B, open arrowhead). Fibromuscular stroma did not stain positive for SphK1. CgA staining was observed in a minority of cancer cells, approximately 10%, in the form of secretory granuli (H, Fig. 8D). Co-expression of CgA in blue with brown SphK1 resulted in intense dark brown signal (solid arrowhead). Note in Fig. 8A (bottom) and Fig. 8C, the neuron-like appearance of the double-labeled cancer cell (solid arrow). The transition to the androgen refractory state during chronic androgen depletion is characterized in vitro by a loss of response of androgen-deprived cells to either FBS or DHT [20]. As early as 7 days and by 14 days of androgen withdrawal, LNCaP were totally unresponsive after transfer to FBS conditions or when treated with PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e8048