EXTRACELLULAR SPHINGOSINE-1-PHOSPHATE: A NOVEL ACTOR IN HUMAN GLIOBLASTOMA STEM CELL SURVIVAL PROPERTIES

Glioblastoma multiforme (GBM) is the most frequent and aggressive intracranial tumour in humans. The prognosis of GBM patients remains unfavourable even after aggressive treatments based on multiple approaches, due to the high proliferation rate, migrating-invasive properties, and resistance to therapeutic intervention. The introduction of the alkylating agent TMZ in glioblastoma therapy has improved patient survival, but drug resistance mechanisms limit its benefits. The aim of this study was to provide a contribution to the understanding of the malignant and chemoresistance properties in GBM by focusing on the role of the bioactive sphingoid molecules ceramide and S1P, which act as antagonists in regulating cell properties and survival. Accumulating literature indicates that ceramide is a tumour suppressor sphingolipid, able to induce antiproliferative and apoptotic responses, and that it is able to act as a major player in the mechanism of action of many chemotherapeutic drugs. We demonstrated that the treatment of T98G human glioblastoma cells with cytotoxic TMZ concentrations results in a significant increase in intracellular ceramide, which in turn promotes cell death. On the other hand, TMZ is not able to induce ceramide accumulation in TMZ-resistant glioblastoma cells (TMZ-R). These data suggest a role of ceramide as a mediator of TMZ-induced toxicity. A large amount of evidence underlines the role of S1P as an important tumour-promoting sphingolipid, acting predominantly in the extracellular milieu after interaction with specific G protein-coupled receptors and exerting opposite effects on cell survival compared to ceramide. Parallel studies demonstrated that S1P secretion in TMZ-R cells is functional to inhibit the cytotoxic effect of ceramide and to confer TMZ-resistant properties to glioblastoma cells. Stimulated by these findings, we next evaluated the role of sphingolipid mediators in the malignant features of glioblastoma stem cells (GSCs), a cell subpopulation within the tumour mass involved in the aberrant expansion and therapy resistance properties of glioblastomas. To this purpose we used GSCs isolated from the human U87-MG glioblastoma cell line and GSCs isolated from a primary culture of human glioblastoma. We found that both GSC models efficiently form typical neurosphere structures in mitogen-defined medium and express high levels of recognized cancer stem cell markers. Moreover, GSCs exhibit resistance to TMZ at concentrations that are cytotoxic in U87-MG, despite not expressing the DNA repair protein MGMT, a major contributor to TMZ-resistance. Even though a large amount of evidence underlines that S1P is able to favor growth, invasion and chemotherapy resistance of glioblastoma cells, so far little is known on the possible role of S1P as a factor modulating GSCs malignant properties. Further experiments revealed that glioblastoma cells and GSCs are able to efficiently synthesize S1P and also to release it in the culture medium. Notably the intracellular S1P level was found much lower in GSC models than in the glioblastoma cell line; meanwhile the extracellular S1P level was significantly higher in GSC models than in U87-MG cells. These differences resulted in an extracellular S1P-intracellular S1P ratio at least 10 times higher in GSCs compared to U87-MG. Furthermore, this ratio is about 1:1 in both GSCs, thus suggesting that these cells are an efficient source of S1P in the extracellular microenvironment. Furthermore we found that ceramide-extracellular S1P ratio is at least 2-fold lower in GSCs than in U87-MG. Since S1P and ceramide exert opposing effects on cell survival, according to the “sphingolipid rheostat” model, this different ratio could promote GSC survival observed after TMZ treatment. Interestingly, enzyme activity assays excluded the presence of sphingosine kinase (SK), the enzyme responsible for S1P byosinthesis, in GSC medium, implicating an efficient secretion of S1P in GSCs. The analyses of the expression of the ABC-transporters known to be involved in S1P export (ABCG2, ABCA1 and ABCC1), revealed that only ABCA1 is expressed in GSCs. Notwithstanding, after ABCA1 inhibition, no variations in S1P release was observed, suggesting that other mechanisms different from those known are involved. We also investigated the role of S1P in glioblastoma resistance to TMZ. A first interesting finding was that exogenously administered S1P protected U87-MG cells against TMZ cytotoxic effects. In addition, we found that, after co-treatment with TMZ and an inhibitor of S1P biosynthesis, GSCs became sensitive to the toxic effect of the drug. Of note, exogenous S1P administration was able to revert this effect. These data strongly support extracellular S1P as an important mediator in TMZ-resistance of GSCs. Furthermore, results obtained in GSCs isolated from two patients affected by glioblastoma with different aggressive phenotype, revealed that the extracellular release of S1P was significantly higher by cells isolated from the most aggressive tumour, suggesting that the release and thus the levels of extracellular S1P might be related to tumour aggressiveness and patient prognosis. In conclusion, our data implicate for the first time GSCs as an important source of S1P in the extracellular microenvironment, where, on its turn, S1P can act as an autocrine/paracrine messenger able to contribute to the GSC survival properties. A better understanding of S1P role in GSCs aggressive phenotype could represent a critical start point that sets the bases for the development of new compounds able to sensitize GSCs to chemotherapeutic treatments, thus improving survival rates in GBM patients.