Abstract 3256: Sphingolipid regulation by sphingosine kinase anchoring protein (SKAP) and its implication in cancer

Abstract

Abstract Background: Sphingolipids are important in cancer cell signalling. Sphingosine 1 phosphate (S1P) promotes cell survival and resistance to apoptosis, while S1P precursors ceramide (CER) and sphingosine (SPH), mediate antiproliferative and apoptotic responses. S1P is generated from SPH by sphingosine kinase (SK) enzymes (SK1 and SK2), with SK activity and localisation regulated by other proteins, including PKC, PKA and a SK anchoring protein (SKAP) that has been reported to negatively regulate SK1 activity in fibroblasts. S1P localisation is thought to play an important role in its function. Based on our preliminary observation in primary AML cells that SKAP expression resulted in an increase in S1P, we have investigated the effect of SKAP transfection on S1P production and localisation. Methods: K562, (and for some confirmatory experiments MCF-7), cells were transfected with the SKAP gene using standard techniques. SKAP is normally silenced in both cell lines. Transfection was confirmed by RNA expression. Intracellular and extracellular S1P and SPH, and intracellular SK activity (based on the production of C17 S1P from C17 SPH, an unnatural SPH that is a SK substrate) in intact cells were measured by LC-MS/MS. Phorbol 12-myristate 13-acetate (PMA) was used to induce membrane associated SK function, and MK-571 and fumitremorgen C (FTC) were used to block S1P efflux through ABCC1 and ABCG2 efflux pumps, respectively. Chemosensitivity to doxorubicin and imatinib in transfected cells was also studied. Results: K562 cells transfected with the SKAP gene showed a 2.5 fold increase in intracellular and extracellular levels of basal S1P compared to vector alone control. (In MCF-7 cells SKAP transfection resulted in an almost 10-fold increase in S1P). Further studies in K562 cells confirmed a significant increase in intracellular SK activity in SKAP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p< 0.05). This increase was also observed, though to a lesser extent, in extracellular C17 S1P (678 ± 50 in SKAP vs 462 ± 47 pg/ml in vector alone, p< 0.05). In a proliferation assay this increase in SK activity was associated with a 25% increase in viable cell number (p<0.01 after 3 days). SK activity could be induced further in SKAP cells by the PKC activator PMA, although to a lesser extent than in vector alone cells, while the addition of MK-571 and FTC resulted in a marked increase in intracellular S1P in SKAP and vector alone cells, with a decrease in extracellular S1P levels. These experiments confirm the membrane localisation of SK1 in SKAP transfected cells. SKAP transfection did not affect sensitivity to imatinib or doxorubicin compared to vector alone. Conclusion: These data suggest that SKAP may act as a positive regulator of SK1 activity in cancer cells, an observation that has implications in carcinogenesis and chemosensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3256. doi:1538-7445.AM2012-3256