prostate cancer progression to androgen independence and poor clinical outcome [10,11,12,13,14]. Numerous studies have shown that, after long-term ADT, prostate cancer cells acquire a neuroendocrine (NE)-like pheno- type leading to tumor populations enriched in NE cells. NE cells constitute a minor component of the normal prostate gland and secrete several neuropeptides that can induce mitogenic effects on adjacent cancer cells in androgen-depleted conditions [15]. Although NE cells have been described decades ago, their functional roles in prostate cancer progression have only recently received considerable attention. Neuroendocrine tumor and serum biomarkers are up-regulated following ADT in prostate cancer patients indicative of a poor prognosis [16,17,18,19]. Consistent to clinical observations, androgen withdrawal-induced NE differen- tiation is also seen in cell culture and animal models [20,21,22,23,24], and the transgenic adenocarcinoma of the mouse prostate model of prostate (TRAMP) cancer shows a marked increase in prostate neuroendocrine cell population with disease progression [25]. Sphingosine 1-phosphate (S1P) is a lipid mediator which plays a major regulatory role in tumor cell growth, survival, invasion, and angiogenesis [26]. The balance between the cellular levels of S1P and its metabolic precursors ceramide and sphingosine is regarded as a switch that could determine whether a cell proliferates or undergoes apoptosis or growth arrest [27]. A key regulator of this balance is the sphingosine kinase-1 (SphK1), the enzyme converting sphingosine into S1P. SphK1 serves the dual function of producing the pro-growth, anti-apoptotic S1P, and decreasing intracellular levels of pro-apoptotic ceramide. Further supporting a role for SphK1 in promoting cancer, SphK1 has been found to act as an oncogene [28], its mRNA is overexpressed and positive immunostaining for SphK1 was found in various tumors [29,30,31,32,33], and the increase in SphK1 expression in tumor biopsies was correlated with short survival rate in patients with glioblastoma and breast cancers [30,34]. In addition, SphK1 enzymatic activity and expression are markedly increased in tumor samples from prostate cancer patients (as compared with normal counterparts) correlating with other markers such as PSA level, tumor grade as well as with the clinical outcome after prostatectomy (Malavaud and Cuvillier, submitted). While SphK1 activity can be stimulated by a wide array of growth factors [26], we have previously shown in prostate cancer cell and animal models that anticancer treatments (chemotherapeutic agents or ionizing radiations) lead to its inhibition suggesting that SphK1 could act a sensor to anticancer therapies [35,36,37,38]. In this study, we explored the potential role of SphK1 in the regulation of androgen-dependent cell growth and survival in the hormone-sensitive LNCaP prostate cancer cell model. For the first time, we show that androgen deprivation exerts a con- trasting effect on SphK1. While short-term androgen withdrawal induced a temporary inhibition of SphK1, chronic androgen depletion triggered an up-regulation of SphK1 correlating with the NE differentiation of LNCaP and C4-2B cells support- ing the involvement of SphK1 in the progression toward androgen-independence. Results Short-Term Androgen Deprivation Decreases Cell Proliferation in LNCaP - but Not in PC-3 Cells - and Is Associated with Decreased SphK1 Activity Cell proliferation was markedly reduced overtime in CSS (androgen-free medium)-treated LNCaP cells when compared to FBS (which contains low levels of androgens) treated cells (Fig. 1A, SphK1 and Androgen Signaling left panel). To support MTT results, cell counting (Fig. 1A, middle 3 panel) and [ H]thymidine incorporation (Fig. 1A, right panel) measurements confirmed that CSS conditions had a dramatic effects on cell number and DNA synthesis establishing that MTT could be used as a surrogate index for cellular proliferation in our cell model. This was also correlated with the secretion of PSA whose level was strongly reduced in both CSS-treated cells as compared to FBS-treated cells (Fig. 1B). On the contrary, androgen deprivation did not alter the growth of PC-3 hormono-refractory (HR) cells whose growth was only altered by serum starvation (Fig. 1C).When compared to FBS conditions, androgen depletion in LNCaP induced a notable decrease in SphK1 activity within the first 24 h (Fig. 1D, left panel). Later, a rebound in SphK1 activity was observed, which became significant beyond 4 days of treatment (Fig. 1D, left panel). In PC-3 cells, a significant and lasting decrease in SphK1 activity was only observed under serum deprivation conditions (Fig. 1D, right panel). mirroring the impact on cell proliferation (Fig. 1C). As anticipated in PC-3 cells that are unaffected by CSS conditions, no significant SphK1 changes could be evidenced (Fig. 1C, D). The Efficacy of Castration in Orthotopically Xenotransplanted SCID Mice Is Associated with SphK1 Inhibition The effect of androgen deprivation was next examined in vivo using a surgical orthotopic implantation (SOI) of LNCaP and PC- 3 cells overexpressing GFP [36]. Nine and three weeks after SOI of LNCaP/GFP and PC-3/GFP cells respectively, SCID mice were randomized into two groups and subjected to castration or sham treatment. As shown by high-magnification microscopy of a representative primary LNCaP/GFP tumor, castration induced a dramatic decrease in tumor volume and mass (Fig. 2A and B) within 7 days of treatment compared to sham-treated animals. The efficacy of castration was associated with a significant decrease in SphK1 activity in tissue extracts (Fig. 2B, right panel). Additionally, the effect on primary tumor was paralleled by a marked reduction in metastasis dissemination in the castration- treated group (Table S1). As expected, castration did not effect tumor development in SCID mice xenotransplanted with PC-3/ GFP cells (Fig. 2C). Tumor masses and volumes were similar in both sham-treated and castrated animals (Fig. 2D), and SphK1 activity (Fig. 2D, right panel) as well as metastasis dissemination (Table S1) were comparable in both groups. SphK1 Overexpression Renders LNCaP Cells Less Sensitive to Androgen Depletion Because an inhibition of SphK1 was observed during short-term androgen deprivation in vitro (Fig. 1D) and in vivo (Fig. 2B), we verified whether transfection of LNCaP cells with SphK1 might render these cells more resistant to androgen depletion. Transfec- tion efficiency was verified by immunoblotting (Fig. 3A, left panel). The SphK1 activity of SphK1-overexpressing LNCaP (Fig. 3A, right panel) was increased to ,1100 pmol/mn/mg protein (i.e., ,30-fold higher compared to that of empty-vector transfected cells). The instrumental role of SphK1 inhibition in reduced cell growth was confirmed by cell viability assays, which showed that LNCaP overexpressing SphK1 were markedly less sensitive to the effects of androgen depletion (Fig. 3B). Concomitantly, SphK1 enforced expression was associated with a higher secretion of PSA in CSS-treated LNCaP cells reflecting its effects on cell proliferation (Fig. 3C). PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e8048