(a, b) PPC1 cells were infected with Ad-GFP, Ad-AC (MOI 50), Ad-shSCR or Ad-shASAH1 (MOI 20) 1 day prior to plating on six-well plates in a total volume of 4?ml soft agar

(a, b) PPC1 cells were infected with Ad-GFP, Ad-AC (MOI 50), Ad-shSCR or Ad-shASAH1 (MOI 20) 1 day prior to plating on six-well plates in a total volume of 4?ml soft agar. prostate tumors, and elevation of phosphorylated Akt in tumor versus patient-matched benign tissue is contingent upon AC elevation. Investigation of the mechanism for AC-induced Akt activation revealed that AC activates Akt through sphingosine kinase 1 (SphK1)-derived generation of S1P. This signaling pathway proceeds through S1P receptor 2 (S1PR2)-dependent stimulation of PI3K. Functionally, AC-overexpressing cells are insensitive to cytotoxic chemotherapy, however, these cells are more susceptible to targeted inhibition of Akt. AC-overexpressing cells proliferate more rapidly than control cells and form more colonies in soft agar; however, these effects are profoundly sensitive to Akt inhibition, demonstrating increased dependence on Akt signaling for the oncogenic phenotypes of AC-overexpressing cells. These observations may have clinical implications for targeted therapy as PI3K and Akt inhibitors emerge from clinical trials. antisense or empty vector (shAC or pLKO.1) were analyzed by western blotting. (c) MIA, Panc01, SCC14A and PPC1 cells were infected with Ad-AC or Ad-GFP. After 48?h of infection, cells were analyzed by western blotting. (d) DU145 cells were infected with Ad-GFP, Ad-AC or Ad-shAC. After 48?h of infection, cells were analyzed by western blotting. (e) PPC1 cells were infected with Ad-AC or Ad-GFP. After 48?h of infection, cells were analyzed by western blotting. pAkt/tAkt is the ratio of phosphorylated Akt to total Akt normalized to the reference (that is, Ad-GFP is the reference of Ad-AC). SphK1 mediates AC-induced Akt activation The bioactive lipids ceramide, sphingosine and S1P have all been linked to the regulation of Akt. We observed no change in total cell ceramide in Ad-AC-infected PPC1 cells compared with Ad-GFP (Figure 3a), though species-specific alterations were observed (data not shown). Sphingosine and S1P were significantly elevated in Ad-AC-infected cells (Figure 3a). In order to measure secreted S1P, we treated Ad-AC/GFP-infected PPC1 cells with C17-C6 ceramide, finding significant C17-S1P increase in the cells (Supplementary Figure 2A) and medium (Supplementary Figure 2B). Treatment of cells with exogenous sphingosine did not activate Akt, rather decreasing pAkt moderately after 6?h of treatment (Figure 3b). Addition of the dual-isoform sphingosine kinase inhibitor SKICII decreased Akt activation at 6?h, and did not augment Akt activation alone or in combination with sphingosine (Figure 3b). We then infected PPC1 cells with Ad-AC or Ad-GFP in the presence of SKICII, and observed a dose-dependent reduction in Akt activation (Figure 3c, Supplementary Figure 1F), suggesting that sphingosine kinase activity is necessary for AC-induced Akt activation. Infection of wild-type (WT) or sphingosine kinase 2-knocked out (SphK2 KO) mouse embryonic fibroblasts (MEFs) with Ad-AC promoted strong activation of Akt, whereas AC had no impact on Akt activation in SphK1 KO MEFs (Figure 3d, Supplementary Figure 1G). Cyclazodone Ad-AC increased S1P cell content (Supplementary Figure 2C) and secretion into the medium (Supplementary Figure 2D) in WT and SphK2 KO MEFs, but not in SphK1 KO MEFs. To confirm the observation that SphK1 may be necessary for AC-induced Akt activation, we used shRNA (Figure 3e, Supplementary Figure 1H) and small-interfering RNA (siRNA) (Figure 3f, Supplementary Figure 1I) to knock down each SphK isoform and confirmed that knockdown of SphK1, but not SphK2, abrogated AC-induced Akt activation. Open in a separate window Figure 3 SphK1 mediates AC-induced Akt activation. (a) Ad-GFP- or Ad-AC-infected PPC1 cell pellets were analyzed by LC/MS for ceramide, sphingosine and S1P. Bars represent sphingolipid level relative to Ad-GFP. * em P /em 0.05 analyzed by Student’s em t /em -test. (b) PPC1 cells were treated for 2?h with the indicated dose of SphK inhibitor SKICII or vehicle (dimethyl sulphoxide, DMSO) before treatment with the indicated doses of sphingosine or vehicle (EtOH). Cells were collected 2 or 6?h after addition of sphingosine, as indicated. pAkt/tAkt ratios were generated using NIH ImageJ band densitometries, and are represented as raw Cyclazodone ratios so that multiple comparisons can be made. (c) PPC1 cells were infected.Further analysis revealed that S1PR2 mRNA is induced slightly (1.3C2 fold), but significantly, upon AC expression (Supplementary Figure 4B), whereas the other ceramidases are not affected by AC expression, except for a reduction in ACER1 mRNA in PPC1 (Supplementary Figure 4C). targeted inhibition of Akt. AC-overexpressing cells proliferate more rapidly than control cells and form more colonies in soft agar; however, these effects are profoundly sensitive to Akt inhibition, demonstrating increased dependence on Akt signaling for the oncogenic phenotypes of AC-overexpressing cells. These observations may have clinical implications for targeted therapy as PI3K and Akt inhibitors emerge from clinical trials. antisense or empty vector (shAC or pLKO.1) were analyzed by western blotting. (c) MIA, Panc01, SCC14A and PPC1 cells were infected with Ad-AC or Ad-GFP. After 48?h of infection, cells were analyzed by western blotting. (d) DU145 cells were infected with Ad-GFP, Ad-AC or Ad-shAC. After 48?h of infection, cells were analyzed by western blotting. (e) PPC1 cells were infected with Ad-AC or Ad-GFP. After 48?h of infection, cells were analyzed by western blotting. pAkt/tAkt LILRB4 antibody is the ratio of phosphorylated Akt to total Akt normalized to the reference (that is, Ad-GFP is the reference of Ad-AC). SphK1 mediates AC-induced Akt activation The bioactive lipids ceramide, sphingosine and S1P have all been linked to the regulation of Akt. We observed no change in total cell ceramide in Ad-AC-infected PPC1 cells compared with Ad-GFP (Figure 3a), though species-specific alterations were observed (data not shown). Sphingosine and S1P were significantly elevated in Ad-AC-infected cells (Figure 3a). In order to measure secreted S1P, we treated Ad-AC/GFP-infected PPC1 cells with C17-C6 ceramide, finding significant C17-S1P increase Cyclazodone in the cells (Supplementary Figure 2A) and medium (Supplementary Figure 2B). Treatment of cells with exogenous sphingosine did not activate Akt, rather decreasing pAkt moderately after 6?h of treatment (Figure 3b). Addition of the dual-isoform sphingosine kinase inhibitor SKICII decreased Akt activation at 6?h, and did not augment Akt activation alone or in combination with sphingosine (Figure 3b). We then infected PPC1 cells with Ad-AC or Ad-GFP in the presence of SKICII, and observed a dose-dependent reduction in Akt activation (Figure 3c, Supplementary Figure 1F), suggesting that sphingosine kinase activity is necessary for AC-induced Akt activation. Infection of wild-type (WT) or sphingosine kinase 2-knocked out (SphK2 KO) mouse embryonic fibroblasts (MEFs) with Ad-AC promoted strong Cyclazodone activation of Akt, whereas AC had no impact on Akt activation in SphK1 KO MEFs (Figure 3d, Supplementary Figure 1G). Ad-AC increased S1P cell content (Supplementary Figure 2C) and secretion into the medium (Supplementary Figure 2D) in WT and SphK2 KO MEFs, but not in SphK1 KO MEFs. To confirm the observation that SphK1 may be necessary for AC-induced Akt activation, we used shRNA (Figure 3e, Supplementary Figure 1H) and small-interfering RNA (siRNA) (Figure 3f, Supplementary Figure 1I) to knock down each SphK isoform and confirmed that knockdown of SphK1, but not SphK2, abrogated AC-induced Akt activation. Open in a separate window Figure 3 SphK1 mediates AC-induced Akt activation. (a) Ad-GFP- or Ad-AC-infected PPC1 cell pellets were analyzed by LC/MS for ceramide, sphingosine and S1P. Bars represent sphingolipid level relative to Ad-GFP. * em P Cyclazodone /em 0.05 analyzed by Student’s em t /em -test. (b) PPC1 cells were treated for 2?h with the indicated dose of SphK inhibitor SKICII or vehicle (dimethyl sulphoxide, DMSO) before treatment with the indicated doses of sphingosine or vehicle (EtOH). Cells were collected 2 or 6?h after addition of sphingosine, as indicated. pAkt/tAkt ratios were generated using NIH ImageJ band densitometries, and are represented as raw ratios so that multiple comparisons can be made. (c) PPC1 cells were infected with Ad-AC or Ad-GFP in the presence of the indicated dose of SKICII. After 48?h of infection, cells were analyzed by western blotting. (d) WT, SphK1 KO and SphK2 KO MEFs were infected with Ad-AC or Ad-GFP at multiplicity of infection (MOI) 100. After 48?h of infection, cells were analyzed by western blotting. (e) PPC1 cells were transfected with nontargeting (SCR) or SphK1, or SphK2-targeting shRNA vectors. After 6?h of transfection, cells were infected with Ad-GFP or Ad-AC. After 48?h of infection, cells were analyzed by western blotting and qRTCPCR. (f) PPC1 cells were transfected with nontargeting (SCR) or SphK1, or Sphk2-targeting siRNA. After 6?h of transfection, cells were infected with Ad-GFP or Ad-AC. After 48?h of infection, cells were analyzed by western blotting and qRTCPCR. S1PR2 stimulates PI3K to activate Akt To determine whether.