We examined the involvement of sphingosine kinase-1 (SphK1), which governs the ceramide/sphingosine-1-phosphate balance, in susceptibility to imatinib of either sensitive or resistant chronic myeloid leukemia cells. Imatinib-sensitive LAMA84-s displayed marked SphK1 inhibition coupled with increased content of ceramide and decreased pro-survival sphingosine-1-phosphate. Conversely, no changes in the sphingolipid metabolism were observed in LAMA84-r treated with imatinib. Overcoming imatinib resistance in LAMA84-r with farnesyltransferase or MEK/ERK inhibitors as well as with cytosine arabinoside led to SphK1 inhibition. Overexpression of SphK1 in LAMA84-s cells impaired apoptosis and inhibited the effects of imatinib on caspase-3 activation, cytochrome c and Smac release from mitochondria through modulation of Bim, Bcl-xL and Mcl-1 expression. Pharmacological inhibition of SphK1 with F-12509a or its silencing by siRNA induced apoptosis of both imatinib-sensitive and -resistant cells, suggesting that SphK1 inhibition was critical for apoptosis signaling. We also show that imatinib-sensitive and -resistant primary cells from chronic myeloid leukemia patients can be successfully killed in vitro by the F-12509a inhibitor. These results uncover the involvement of SphK1 in regulating imatinib-induced apoptosis and establish that SphK1 is a downstream effector of the BcrAbl/Ras/ERK pathway inhibited by imatinib but upstream regulator of Bcl-2 family members.

Sphingosine 1-phosphate (S1P) plays important roles in cell proliferation, differentiation or survival mainly through its surface G-protein-coupled receptors S1P1-5. Bone represents the major site of metastasis for prostate cancer (CaP) cells, which rely on bone-derived factors to support their proliferation and resistance to therapeutics. In the present work we have found that conditioned medium (CM) from the MC3T3 osteoblastic cell line or primary murine and human osteoblast-like cells, as well as co-culture with MC3T3 stimulate proliferation of CaP lines in S1P-dependent manner. In addition, osteoblastic-derived S1P induces resistance of CaP cells to therapeutics including chemotherapy and radiotherapy. When S1P release from osteoblastic cells is decreased (inhibition of SphK1, knock-down of SphK1 or the S1P transporter, Spns2 by siRNA) or secreted S1P neutralized with anti-S1P antibody, the proliferative and survival effects of osteoblasts on CaP cells are abolished. Because of the paracrine nature of the signaling, we studied the role of the S1P receptors expressed on CaP cells in the communication with S1P secreted by osteoblasts. Strategies aimed at down-regulating S1P1, S1P2 or S1P3 (siRNA, antagonists), established the exclusive role of the S1P/S1P1 signaling between osteoblasts and CaP cells. Bone metastases from CaP are associated with osteoblastic differentiation resulting in abnormal bone formation. We show that the autocrine S1P/S1P3 signaling is central during differentiation to mature osteoblasts by regulating Runx2 level, a key transcription factor involved in osteoblastic maturation. Importantly, differentiated osteoblasts exhibited enhanced secretion of S1P and further stimulated CaP cell proliferation in a S1P-dependent manner. By establishing the dual role of osteoblast-borne S1P on both osteoblastic differentiation and CaP cell proliferation and survival, we uncover the importance of S1P in the bone metastatic microenvironment, which may open a novel area of study for the treatment of CaP bone metastasis by targeting S1P.