Sphingosine Kinase as an Oncogene: Autocrine Sphingosine 1-Phoshate Modulates ML-1 Thyroid Carcinoma Cell Migration by a Mechanism Dependent on Protein Kinase C-α and ERK1/2

CANCER-ONCOGENES Sphingosine Kinase as an Oncogene: Autocrine Sphingosine 1-Phoshate Modulates ML-1 Thyroid Carcinoma Cell Migration by a Mechanism Dependent on Protein Kinase C-␣ and ERK1/2 N. Bergelin, T. Blom, J. Heikkilä, C. Löf, C. Alam, S. Balthasar, J. P. Slotte, A. Hinkkanen, and K. Törnquist Departments of Biology (Cell Biology) (N.B., C.L., C.A., S.B., K.T.) and Biochemistry and Pharmacy (J.H., J.P.S., A.H.), Åbo Akademi University, and Turku Graduate School of Biomedical Sciences (N.B.), 20520 Turku, Finland; The Minerva Foundation Institute for Medical Research (N.B., K.T.), 00290 Helsinki, Finland; and Institute of Biomedicine/Anatomy (T.B.), University of Helsinki, 00250 Helsinki, Finland Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P1 and S1P3 receptors, Gi proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C␣, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C␣, ERK1/2, and SK. (Endocrinology 150: 2055–2063, 2009) S phingosine 1-phosphate (S1P) has been implicated in a variety of different cellular events and couples to central biological processes, such as development, immunology, and the progression of cancer (1– 4). Extracellular S1P signals through five high-affinity G protein-coupled receptors, targeting different intracellular cascades (5). In addition, S1P signaling can be mediated through a yet-unidentified intracellular mechanism (6, 7). S1P is considered a tumor-promoting agent because it regulates growth, adhesion, migration, angiogenesis, metastasis, and survival in cancer (4). A sphingolipid rheostat has been proposed, in which opposite effects of apoptotic sphingosine and ceramide signaling and prosurvival S1P signaling can tip the cell fate trough the actions of sphingosine kinase (SK) (8). Thus, there have been attempts to target S1P signaling in cancer therapy to favor ceramide and sphingosine production and the breakdown of S1P. Novel potential therapies include the targeting of S1P receptors with the immunosuppressant FTY720 (9) and the development of potent SK inhibitors (10). Furthermore, the development of an anti-S1P antibody has opened possibilities for more targeted cancer treatment (11). S1P is produced by the phosphorylation of sphingosine by two known isoforms of SK, differing in cellular location, expression during development, and function (reviewed in Ref. 12). The two SKs can be activated by a multitude of effectors, such as ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2008-0625 Received April 30, 2008. Accepted December 22, 2008. First Published Online December 30, 2008 Abbreviations: ABCC1, ATP-binding cassette transporter C1; FCS, fetal calf serum; HBDDE, 2,2⬘,3,3⬘,4,4⬘-hexahydroxy-1,1⬘-biphenyl-6,6⬘-dimethanol dimethyl ether; HPTLC, highperformance thin-layer chromatography; LS-FBS, lipid-stripped-fetal bovine serum; OAG, 1-oleyl-2-acetyl-sn-glycerol; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; Ptx, pertussis toxin; SFM, serum-free medium; siRNA, small interfering RNA; SK, sphingosine kinase; SKi, SK inhibitor; S1P, sphingosine 1-phosphate; WT, wild type. Endocrinology, May 2009, 150(5):2055–2063 endo.endojournals.org 2055 2056 Bergelin et al. Sphingosine Kinase, S1P, and Migration growth factors and cytokines (reviewed in Ref. 13). SK may be regulated by protein-protein interactions, phosphorylation, Ca2⫹, and subcellular location (12), and it is still unclear whether all or only combinations of these mechanisms are required for the full activation of SK (13). Using tumor growth in immunodeficient mice and the colony formation of cells on soft agar (14), the oncogenic effect of SK has been elucidated. Recently a high SK expression was identified as a marker for poor prognosis and increased metastasis of human breast cancer (15). In addition, SK overexpression studies in NIH 3T3 cells confirmed the progrowth function of SK because the G1/S cell cycle transition and the production of S1P was promoted (16), suggesting that the effects of overexpression of SK may be due to an autocrine effect by S1P (13). An ATP-binding cassette transporter [ATP-binding cassette transporter C1 (ABCC1)] has been identified as, at least in part, being responsible for the secretion of S1P (17). The secreted S1P may subsequently activate S1P receptors in an autocrine or paracrine manner. Recently an ATP-binding cassette transporter A1 (ABCA1) transporter was also identified to be involved in S1P secretion in astrocytes (18). In the present study, we examined the role of human SK in the migration, proliferation, and adhesion of the human thyroid follicular carcinoma cell line ML-1. Overexpression of SK enhances the serum-induced migration of ML-1 cells in an ABCC1-dependent manner, suggesting autocrine S1P signaling. Inhibiting protein kinase C (PKC)-␣, with both small interfering RNA (siRNA) and small molecular inhibitors, attenuates migration in SK-overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and up-regulation of ERK1/2 phosphorylation, compared with cells expressing a dominant-negative SK, SKG82D (19). Materials and Methods Materials DMEM, Coon’s F12 medium, horseradish peroxidase-conjugated goat antimouse, anti-FLAG M2 antibody, N-TER nanoparticle siRNA transfection system, doxorubicin, D-erythro-sphingosine, 1-oleyl-2acetyl-sn-glycerol (OAG), pertussis toxin (Ptx), insulin, transferrin, hydrocortisone, tripeptide gly-L-his-L-lys, somatostatin, and fatty acid-free BSA, and BSA were purchased from Sigma (St. Louis, MO). Fetal calf serum (FCS), Ham’s F-12 medium, penicillin/streptomycin, trypsin, and L-glutamine were from Invitrogen (Carlsbad, CA). Cell culture plasticw (...truncated)