S1P1 and VEGFR-2 form a signaling complex with extracellularly regulated kinase 1/2 and protein kinase C-alpha regulating ML-1 thyroid carcinoma cell migration.

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor receptor 2 (VEGFR-2) signaling have been shown to integrate in many biological processes. The follicular thyroid carcinoma cell line ML-1 expresses VEGFR-2 and secretes substantial amounts of both vascular endothelial growth factor (VEGF)-A and VEGF-C. ML-1 cells also express S1P-receptors (S1P(1-3,5)). S1P is able to phosphorylate VEGFR-2, and inhibiting VEGFR-2 attenuates S1P-induced migration and down-regulates S1P(1) expression in ML-1 cells. In the present study, we focused on the interactions between S1P(1) and VEGFR-2. We show that S1P receptors form complexes with VEGFR-2 and that the S1P(1)/VEGFR-2 complex associates with protein kinase C (PKC)-alpha and ERK1/2. Furthermore, the complex evokes bidirectional signaling since the S1P-induced ERK1/2 phosphorylation is sensitive to VEGFR-2 kinase inhibition and VEGF-A-induced ERK1/2 phosphorylation is sensitive to pertussis toxin treatment as well as S1P(1) small interfering RNA (siRNA) treatment. Both S1P- and VEGF-A-induced haptotaxis is sensitive to pertussis toxin treatment and S1P(1) siRNA treatment. Phosphorylation of ERK1/2 evoked by both VEGF-A and the S1P(1) agonist SEW-2871 is inhibited by PKC-alpha and PKC-betaI siRNA. We hypothesize that VEGFR-2 forms a signaling complex with S1P(1), evoking bidirectional signaling regulating both ERK1/2 phosphorylation and haptotaxis of ML-1 cells.

Interactions between sphingosine-1-phosphate and vascular endothelial growth factor signalling in ML-1 follicular thyroid carcinoma cells.

Sphingosine-1-phosphate (S1P) induces migration of human ML-1 thyroid follicular cancer cells and inhibits migration of human FRO anaplastic thyroid cancer cells. As tumour cells often secrete vascular endothelial growth factor (VEGF), we investigated a possible interaction between S1P and VEGF signalling in the regulation of thyroid tumour cell migration. We found that both ML-1 and FRO cells secreted VEGF-A ( approximately 3.6 and <0.1 ng/10(6) cells/day respectively) and VEGF-C ( approximately 3.0 and 0.14 ng/10(6) cells/day respectively). S1P stimulated VEGF-A secretion in both cell lines, and blocking S1P receptors 1, 2 and 3 attenuated the S1P-evoked secretion of VEGF-A. Neither TSH nor insulin affected the amount of secreted VEGF-A or -C in ML-1 cells, while simultaneous stimulation with insulin and S1P increased VEGF-C secretion in FRO cells. Both cell lines expressed VEGF receptor 2 (VEGFR-2) mRNA and proteins. Serum-evoked migration of both ML-1 and FRO cells was attenuated when VEGFR-2 was inhibited. Moreover, inhibiting VEGFR-2 in ML-1 cells resulted in a rapid downregulation of S1P1 mRNA expression and S1P1 protein levels, suppression of S1P-induced migration and a decrease in S1P-induced Akt phosphorylation. A VEGF-neutralizing antibody also reduced S1P-induced migration. In ML-1 cells, S1P phosphorylated VEGFR-2. In addition, VEGFR-2 inhibition resulted in the upregulation of S1P3 mRNA within 24 h, but a significant increase in S1P3 protein levels was not observed. VEGFR-2 inhibition, but not a VEGF-neutralizing antibody, reduced ML-1 cell proliferation independently of S1P stimulation. The results indicate a complex interaction between S1P and VEGFR-2 in ML-1 cells, particularly in regulating migratory responses.

Antiproliferative effect of sphingosylphosphorylcholine in thyroid FRO cancer cells mediated by cell cycle arrest in the G2/M phase.

Among the group of bioactive sphingolipids, sphingosylphosphorylcholine (SPC) has been known to induce both antiproliferative and proliferative effects depending on cell type. In the present investigation we show that SPC (1-10 microM) reduced the proliferation of FRO cells (an anaplastic thyroid carcinoma cell line) in a concentration dependent manner. The effect was pertussis toxin insensitive, and independent of phospholipase C, protein kinase C, p38 kinase, or jun kinase. In addition to inhibiting the migration of FRO cells, application of SPC induced a rapid (<10 min) rounding of the cells, which was dependent on extracellular sodium. However, DAPI staining and caspase-3 analysis could not reveal any apoptotic effects of SPC. Furthermore, when cells treated with SPC for 24h were washed and replated, they continued to grow, albeit somewhat slower than control cells. Flow cytometry analysis revealed a significant increase in the population of cells in the G2-M phase, and a reduction in S phase. SPC reduced the phosphorylation of Akt with about 50% and evoked a substantial decrease in the amount of phosphorylated mitogen-activated protein (MAP) kinase. In cells treated with the PI3 kinase inhibitor wortmannin, both migration and proliferation were inhibited, as well as the amount of phosphorylated MAP kinase. Treatment of the cells with either SPC or wortmannin increased the levels of p21, but decreased that of cyclin B1 and Cdc2. Taken together, SPC is an effective suppressor of thyroid cancer cell proliferation and migration, and this effect is, in part, mediated by inhibition of both the PI3K-Akt and the MAP kinase signalling pathways.

Sphingosine 1-phosphate receptor expression profile and regulation of migration in human thyroid cancer cells.

S1P (sphingosine 1-phosphate) receptor expression and the effects of S1P on migration were studied in one papillary (NPA), two follicular (ML-1, WRO) and two anaplastic (FRO, ARO) thyroid cancer cell lines, as well as in human thyroid cells in primary culture. Additionally, the effects of S1P on proliferation, adhesion and calcium signalling were addressed in ML-1 and FRO cells. All cell types expressed multiple S1P receptors. S1P evoked intracellular calcium signalling in primary cultures, ML-1 cells and FRO cells. Neither proliferation nor migration was affected in primary cultures, whereas S1P partly inhibited proliferation in ML-1 and FRO cells. Low nanomolar concentrations of S1P inhibited migration in FRO, WRO and ARO cells, but stimulated ML-1 cell migration. Consistently, S1P1 and S1P3, which mediate migratory responses, were strongly expressed in ML-1 cells, and S1P2, which inhibits migration, was the dominating receptor in the other cell lines. The migratory effect in ML-1 cells was mediated by G(i) and phosphatidylinositol 3-kinase. Both S1P and the S1P1-specific agonist SEW-2871 induced Akt phosphorylation at Ser473. However, SEW-2871 failed to stimulate migration, whereas the S1P1/S1P3 antagonist VPC 23019 inhibited S1P-induced migration. The results suggest that aberrant S1P receptor expression may enhance thyroid cancer cell migration and thus contribute to the metastatic behaviour of some thyroid tumours.