SphK1 and Androgen Signaling Figure 6. The progression of LNCaP to androgen-refractory state is associated with increase in SphK1 expression and activity. A, representative phase-contrast images of LNCaP cells at the beginning of the experiment (Day 0) and after 42 days of incubation in charcoal stripped conditions (Day 42). Chromogranin A (CgA) content was evaluated by immunoblotting at the indicated times (bottom).Secreted Neuron Specific Enolase (NSE) level was measured in culture media from LNCaP cells at the indicated times (right panel). Columns, mean of five independent experiments; bars, SD. The two-tailed P values between the means are as follow: ***, P,0.001. SphK1 activity (B, left panel) was determined at the indicated times in LNCaP cells incubated in CSS conditions. Points, mean of five independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001. Inset, S1P content as shown by TLC. Right panel, expression of SphK1, phospho-Akt, and Akt were analyzed by Western blotting at the indicated times. Similar results were obtained in five independent experiments. C, expression of CgA and phospho-Akt (left panel) and SphK1 activity (right panel) were analyzed 10 days after treatment or not with 1 mM LY294002 or 200 nM wortmannin (WT) under charcoal stripped conditions. Columns, mean of five independent experiments; bars, SD. The two-tailed P values between the means are as follow: ***, P,0.001. D, secreted NSE level was measured in culture media 10 days after treatment or not with 5 mM SKI-2 under charcoal stripped conditions (left panel). Expression of CgA and phospho-Akt were analyzed by western blot (right panel). Columns, mean of five independent experiments; bars, SD. The two- tailed P values between the means are as follow: **, P,0.01. doi:10.1371/journal.pone.0008048.g006 stream of the PI3K/Akt signaling since the canonical PI3K inhibitor, wortmannin could totally abrogate SphK1 stimulation upon DHT addition while SphK1 inhibition had no effect on DHT-induced phosphorylation of Akt. Further supporting the instrumental role played by SphK1 in the adaptation to hormonal environment, the pharmacological SphK1 inhibitors could potently (although not completely) inhibit the increase in cell proliferation and PSA secretion triggered by DHT. Differential mechanisms are observed between short- and long- term androgen deprivation. It is well established both in cell culture and from clinical studies that the transition to the hormone- refractory state is correlated by the acquisition of a neuro-endocrine (NE) like phenotype [16,17,18,19,20,21,22,23,24], in which the activation of the PI3K/Akt signaling has been implicated [5,6,7,8,9,10,11,12,13,14]. A major finding of our study is the spectacular increase in SphK1 activity, which appears to be dependent on cyclic AMP production, during the NE-differentiation process. It is tempting to speculate that the SphK1 activation upon chronic androgen deprivation may serve as an adaptive mechanism allowing prostate cancer cells to survive in androgen-depleted environment, as SphK1 is a well described pro-survival pathway via the effects of sphingosine 1-phosphate [27]. Consistent with an hypothesis of an adaptive functionality for SphK1, replenishing the media with complete serum was associated with the reversal of SphK1 activity towards its basal levels. In addition, pharmacological inhibition of SphK1 during chronic androgen depletion was capable of reversing at least partially the NE differentiation process. Further supporting this uncovered role for SphK1 during the transition to the hormone-refractory state, immunohistochemical studies of prostate cancer human samples showed for the first time that CgA positive-NE cells co-expressed SphK1. As NE cells secrete several neuropeptides with mitogenic effects on adjacent cancer cells in androgen-depleted conditions [15], it would be interesting to determine whether NE cells might represent a source of sphingosine 1-phosphate, which then could act in a paracrine PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e8048