Sphingosine-1 Phosphate (S1P) Is a Myeloma Survival Factor and Protects Myeloma Cells from Thalidomide-Induced Growth Inhibition by Counteracting Ceramide Activity.

Sphingosine 1-phosphate (S1P) and ceramide are sphingolipid metabolites and their intracellular balance impacts fate of normal and cancer cells; whereas S1P stimulates tumor cell migration, survival, proliferation and drug resistance, ceramide acts as endogenous pro-apototic factor. The role of S1P/ceramide pathway in myeloma has not been reported. Recent study demonstrated that thalidomide’s antiangiogenic activity involves increased production of ceramide in microvessels (Yabu et al., Blood 2005). In this study we tested the effect of S1P and ceramide on myeloma growth and determined the protective role of S1P against the inhibitory effect of thalidomide on myeloma cells. S1P (0.1–10 μM) stimulated growth of several human myeloma cell lines in serum-free media in time and dose dependent manners, reaching maximum response (>25%, p<0.001) at approximately 5 μM. Annexin V/PI flow analysis and TUNEL staining for detection of apoptosis revealed reduced percent of apoptotic myeloma cells following S1P treatment. S1P also consistently and significantly protected primary CD138-selected myeloma cells (n=5) from serum starvation-induced apoptosis as shown by MTT assay (p<0.01) and annexin V/PI flow cytometry (p<0.05). In contrast, synthetic ceramide (3–10 μM) inhibited growth of myeloma cells, an effect that was completely counteracted by S1P. We have previously demonstrated that osteoclasts and megakaryocytes support primary myeloma cell survival. Autotaxin, a key exo-enzyme involved in extracellular production of S1P and lysophosphatidic acid (LPA), was consistently upregulated in primary myeloma cells by 11±3 folds over baseline levels after co-culture with osteoclasts (n=17, p<0.01) and by 4±1 folds after co-culture with megakaryocytes (n=5, p<0.04), as assessed by microarray and confirmed by qRT-PCR. Furthermore, in co-cultures the S1P receptors antagonist, FTY720, at concentrations that have no direct anti-tumor response (≤1 μM), inhibited the stimulatory effects of osteoclasts and megakaryocytes on survival of our BN-stroma dependent myeloma cell line by 30% to 50% (p<0.001) suggesting that S1P is an important microenvironmental factor in myelomatous bone. Since metabolic activation of thalidomide is required for its anti-tumor activity, thalidomide (10–100 μM) was incubated with active human microsomes while control non-metabolized thalidomide was similarly prepared using inactivated microsomes. Metabolized but not non-metabolized thalidomide effectively inhibited growth of myeloma cells by >40% (p<0.001). Addition of S1P (1 μM) completely abrogated the inhibitory effect of metabolized thalidomide in all tested doses. We conclude that S1P is a myeloma survival and growth factor and protects myeloma cells from thalidomide-induced growth inhibition, presumably through counteracting increased ceramide activity by thalidomide. Pharmacological modulation of enzymes involved in regulation of the ceramide to S1P ratio and blocking S1P activity could constitute novel therapeutic approaches for treating and overcoming drug resistance in myeloma.