Merkel cell carcinoma (MCC) is an aggressive metastatic neuroendocrine skin cancer associated with high recurrence rates as well as regional and distant metastasis resulting in a high mortality. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid playing a decisive role in promoting tumor cell survival, proliferation, migration and metastasis via intracellular and extracellular targets. SPHK1 and 2 are the enzymes that phosphorylate pro-apoptotic sphingosine to form pro-survival S1P and are upregulated in different cancer entities. The objective of this study was to determine the importance of intracellular S1P in MCC cell survival and proliferation. The SPHK1 and 2 expressions were analyzed by qRT-PCR in 16 MCC cell lines. The well characterized MCC cell lines MKL1 and WaGa were used for functional studies in vitro. Since the cell of origin of MCC is still controversial, normal human dermal fibroblasts (NHDF) were used as non-transformed control cells. Treatment with SKI-II, an inhibitor of SPHK1 and 2, on MKL1 and WaGa resulted in dose dependent decrease of cell viability and increased apoptosis. In contrast, we did not see any effect of SKI-II inhibitor on NHDF cell viability as determined by CellTiter-Glo 3D cell viability assay and FITC Annexin V FACS analysis. Further, S1P-ELISA showed a decrease in intracellular S1P concentration and LC/MS analysis revealed an increase in ceramide and decrease in sphingomyelin species upon treatment with SKI-II in both MKL1 and WaGa cell lines. In addition, immunoblotting revealed decrease in pAKT levels and increased Caspase3 and PARP cleavage in SKI-II treated MKL1 and WaGa. In conclusion our in vitro data suggest that targeting the S1P axis represents a potential therapeutic strategy in MCC.

Interference with sphingosine 1-phosphate synthesis affects Merkel cell carcinoma proliferation in vitro