Bioactive lipids S1P and C1P are prometastatic factors in human rhabdomyosarcoma, and their tissue levels increase in response to radio/chemotherapy

Abstract

Evidence suggests that bioactive lipids may regulate pathophysiologic functions such as cancer cell metastasis. Therefore, we determined that the bioactive lipid chemoattractants sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) strongly enhanced the in vitro motility and adhesion of human rhabdomyosarcoma (RMS) cells. Importantly, this effect was observed at physiologic concentrations for both bioactive lipids, which are present in biologic fluids, and were much stronger than the effects observed in response to known RMS prometastatic factors such as stromal derived factors-1 (SDF-1/CXCL12) or hepatocyte growth factor/scatter factor (HGF/SF). We also present novel evidence that the levels of S1P and C1P were increased in several organs after γ-irradiation or chemotherapy, which indicates an unwanted prometastatic environment related to treatment. Critically, we found that the metastasis of RMS cells in response to S1P can be effectively inhibited in vivo with the S1P-specific binder NOX-S93 that is based on a high-affinity Spiegelmer. These data indicate that bioactive lipids play a vital role in dissemination of RMS and contribute to the unwanted side effects of radio/chemotherapy by creating a prometastatic microenvironment.

Figure 1. Bioactive lipids S1P and C1P are chemoattractants for RMS cells at concentrations corresponding to physiological concentrations in plasma

Panel A - Chemotaxis of RH30 cells in response to S1P (1 μM, considered as physiological dose (13)), C1P (0.5 μM, considered as physiological dose (13)), SDF1 (5 or 300 ng/ml), and HGF (0.3 or 10 ng/ml). Data are pooled from three independent experiments. Panel B - Dose-dependent effect of S1P and C1P on migration of RH30. Panel C - Chemotaxis of different RMS cell lines across transwell membranes in response to S1P (1 μM) or C1P (0.5 μM). Panel D -Chemotaxis and chemokinesis of RH30 and RD cells in response to S1P (1 μM) and C1P (0.5 μM). The chemotaxis assay was done at least twice in duplicate, with similar results. Results are presented as means +/− SD, with a statistical significance relative to the control of *p < 0.05 and **p < 0.01.

Figure 2. S1P and C1P activate MAPK and AKT intracellular pathway proteins and induce migration of human RMS cell lines through G protein-coupled receptors

Panel A - Phosphorylation of MAPK p42/44 and AKT in human RMS cell lines stimulated for 5 min by S1P (0.1, 0.5, or 1 μM) or C1P (0.5 or 1 μM). The experiment was repeated three times with similar results, and a representative study is shown. Panel B – The effect of LY294002, UO126, MK2206 and pertusis toxin (PTX) or vehicle alone on the migration of RH30 and RD cells in response to S1P (1 μM) or C1P (0.5 μM). The experiment was done twice with similar results. *p < 0.05 or **p < 0.01 indicates statistical significance relative to the control (cells migrating in response to S1P or C1P alone). Panel C – qRT-PCR for S1PR_1–3 revealed differences in expression of these receptors between ARMS and ERMS cell lines. The experiment was repeated twice on two different batches of cells, with similar results. Panel D - Chemotaxis of RMS cell lines to S1P (1 μM) in the absence or presence of the S1PR1 inhibitor W146, the S1PR2 inhibitor JTE-013, or the S1PR3 inhibitor BML241. Chemotaxis of cells in the presence of vehicle or inactive analog of W146 – W140 are also presented. The results from two independent experiments are shown as means ± SD. *p < 0.05 or **p < 0.01 compared with the control (cells stimulated with S1P alone).

Figure 2. S1P and C1P activate MAPK and AKT intracellular pathway proteins and induce migration of human RMS cell lines through G protein-coupled receptors

Panel A - Phosphorylation of MAPK p42/44 and AKT in human RMS cell lines stimulated for 5 min by S1P (0.1, 0.5, or 1 μM) or C1P (0.5 or 1 μM). The experiment was repeated three times with similar results, and a representative study is shown. Panel B – The effect of LY294002, UO126, MK2206 and pertusis toxin (PTX) or vehicle alone on the migration of RH30 and RD cells in response to S1P (1 μM) or C1P (0.5 μM). The experiment was done twice with similar results. *p < 0.05 or **p < 0.01 indicates statistical significance relative to the control (cells migrating in response to S1P or C1P alone). Panel C – qRT-PCR for S1PR_1–3 revealed differences in expression of these receptors between ARMS and ERMS cell lines. The experiment was repeated twice on two different batches of cells, with similar results. Panel D - Chemotaxis of RMS cell lines to S1P (1 μM) in the absence or presence of the S1PR1 inhibitor W146, the S1PR2 inhibitor JTE-013, or the S1PR3 inhibitor BML241. Chemotaxis of cells in the presence of vehicle or inactive analog of W146 – W140 are also presented. The results from two independent experiments are shown as means ± SD. *p < 0.05 or **p < 0.01 compared with the control (cells stimulated with S1P alone).

Figure 3. S1P and C1P increase the adhesivness of RMS cells and induce F-actin formation and translocation of paxillin to focal adhesion sites

Panel A - Adhesion of RMS cells to fibronectin. The cells were not stimulated (control) or stimulated with S1P (0.1 or 1 μM) or C1P (0.5 or 1 μM) for 1 h. The number of adherent cells was measured by microscopic analysis. Data from three separate experiments are pooled together and means ± SD are shown. *p < 0.05 and **p < 0.01 compared with the control. Panel B - Adhesion of RH30 and RD cells to mouse stromal cells. RMS cells stained with calcein were stimulated with S1P (1 μM) or C1P (0.5 μM) for 1 h. After a 15-minute incubation, non-adherent cells were removed and adherent cells counted under a fluorescent microscope. Panel C - Actin cytoskeleton organization and paxillin localization in RH30 and RD cells in medium alone or after 4-h stimulation with 1 μM S1P or 0.5 μM C1P. Representative images are shown.

Figure 4. Downregulation of S1PR₁ inhibits RH30 cell growth in vivo and in metastasis

Panel A - qRT-PCR for S1PR₁ in RH30 cells transfected with plasmids encoding shRNA against Renilla (control) and against S1PR₁. The experiment was repeated twice on three different batches of cells with similar results. **p < 0.01. Panel B – FACS analysis of S1PR₁ expression in RH30-shRenilla and RH30-shS1PR₁ cells. The experiment was repeated three times with similar results. **p < 0.01. Panel C - Chemotaxis of RH30 cells transfected with plasmid encoding shRNA against Renilla (control) and against S1PR₁ across Transwell membranes in response to S1P (1 μM). The results from three independent experiments are shown as means ± SD. *p < 0.05 or **p < 0.01 compared with the control. Panel D - RH30 tumor formation after downregulating S1PR₁ expression. Tumor formation by RH30-shRenilla (control), and RH30-shS1PR₁ cells inoculated into the hind limb muscles of SCID/Beige inbred mice. Five weeks later, mice were sacrificed and femora harvested to evaluate the size of the growing tumor. **p<0.01 compared with the control. Panel E - Detection of human RMS cells in bone marrow (BM), liver, lung, and peripheral blood (PB) by qRT-PCR. The results are shown as means ± SD. *p < 0.05 or **p < 0.01.

Figure 5. S1P and C1P levels create a pro-metastatic microenvironment in irradiated organs

Panel A – Conditioned media (CM) from irradiated BM, liver, and lung cells enhance migration of RH30 cell lines across Transwell membranes. The results from three independent experiments are shown as means ± SD. *p < 0.05 or **p < 0.01 compared with the control (CM from cells from non-treated animals). Panel B - Differences in migration in response to CM between control (shRenilla) and cells with downregulation of S1PR1 (shS1PR1). Panel C - Detection of human RMS cells in organs after irradiation or DOP administration. In the experiment, five mice were employed per group. The results are shown as means ± SD. *p < 0.05 or **p < 0.01.

Figure 6. NOX-S93 inhibits S1P-dependent metastasis of RMS to irradiated organs

Panel A – NOX-S93 inhibits dose-dependently the migration of control (shRenilla) RMS cells induced by conditioned medium (CM) from irradiated BM (1500 cGy), whereas it has no effect on RMS cells with knockdown of S1PR1 (shS1PR1). The results from three independent experiments are shown as means ± SD. *p < 0.05 or **p < 0.01 compared with the control (CM from cells from non-treated animals). Panel B - Detection of human RMS cells in organs after irradiation with or without administration of NOX-S93. The results are shown as means ± SD. *p < 0.05 or **p < 0.01 compared with the control. Panel C – Schematic representation of the S1P and C1P effect on RMS metastasis.