A. MAITI1, K. Takabe1, N. C. Hait1 1Roswell Park Cancer Institute,Division Of Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA
Introduction: Cell proliferation and migration are essential components of cancer progression. Mechanism responsible for highly proliferative cancer cells can be specific targets for the treatments of cancer patients. PI3K/AKT/mTOR signaling pathways are involved in crosstalk with RAS/RAF/MEK/ERK and p38 MAPK pathways, which are considered the master controller of proliferation, migration and cell survival. A bioactive sphingolipid signaling mediator sphingosine-1-phosphate (S1P) has emerged as a key regulatory molecule in cancer progression. S1P is generated intracellularly by two isoenzymes, SphK1 and SphK2. Compared to cytosolic SphK1, SphK2 is mainly localized in the nucleus in many cells. S1P is critically involved in the major pathways in oncogenesis including promotion of cellular survival, proliferation and transformation, prevention of apoptosis, stimulation of angiogenesis and metastasis. Here we hypothesized that SphKs/S1P activate ERK, p38 MAP kinases, and PI3K/AKT/mTOR pathways to regulate cancer cell proliferation and migration.
Methods: Experiments were done in human breast cancer cells, MCF7 and MDA-MB-453 cells. Serum starved cell were treated with epidermal growth factor (EGF) and S1P. Endogenous activities of SphKs was inhibited with N,N-Dimethylsphingosine (DMS) and either of SphK1 or SphK2 were ectopically over expressed and treated with EGF. Western blots analysis were done to examine protein expression. Cell migration assay was done by DMS pretreatment followed by EGF and S1P treatment. In order to downregulate one particular SphK, we used specific siRNAs in breast cancer cells and treated with chemotherapeutic drug, doxorubicin to measure cell proliferation, apoptosis, and ceramide levels.
Results: Treatment of cells with S1P from outside activates S1PRs, enhances phosphorylation of ERK, p38, AKT and p70 S6 Kinase1 (SK6). Interestingly, ectopic expression of SphK1 drastically enhanced basal as well as EGF-mediated phosphorylation of those signaling molecules in cultured cells. Down regulation of SphK2 with siRNA, reduced EGF-mediated SphK2 activity and drastically reduced basal as well as EGF-mediated cell proliferation. Further, inhibiting SphKs activities with DMS reduced S1P mediated P-ERK, P-AKT levels. In addition, DMS significantly reduced basal as well as EGF and S1P-mediated cell migration. Downregulation of individual SphK with specific siRNA, enhanced ceramide levels and sensitizes breast cancer cells to doxorubicin mediated apoptosis.
Conclusion: Our data strongly suggested that SphK1, SphK2 and generated S1P are playing critical roles to regulate AKT, ERK and p38 signaling pathways to regulate cancer cell proliferation and migration by using different mechanisms based on their cellular location. This study warrants immediate attention to develop specific inhibitors of each SphK for therapy.