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. 2011 Nov;9(11):1509-19.
doi: 10.1158/1541-7786.MCR-11-0336. Epub 2011 Sep 6.

Ablation of Sphingosine kinase-2 Inhibits Tumor Cell Proliferation and Migration

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Free PMC article

Ablation of Sphingosine kinase-2 Inhibits Tumor Cell Proliferation and Migration

Peng Gao et al. Mol Cancer Res. .
Free PMC article

Abstract

Sphingosine kinases (SK) regulate the balance between proapoptotic ceramides and mitogenic sphingosine-1-phosphate (S1P); however, the functions of the two isoenzymes (SK1 and SK2) in tumor cells are not well defined. Therefore, RNA interference was used to assess the individual roles of SK1 and SK2 in tumor cell sphingolipid metabolism, proliferation, and migration/invasion. Treatment of A498, Caki-1, or MDA-MB-231 cells with siRNAs specific for SK1 or SK2 effectively suppressed the expression of the target mRNA and protein. Ablation of SK1 did not affect mRNA or protein levels of SK2 and reduced intracellular levels of S1P while elevating ceramide levels. In contrast, ablation of SK2 elevated mRNA, protein, and activity levels of SK1 and increased cellular S1P levels. Interestingly, cell proliferation and migration/invasion were suppressed more by SK2-selective ablation than by SK1-selective ablation, showing that the increased S1P does not rescue these phenotypes. Similarly, exogenous S1P did not rescue the cells from the antiproliferative or antimigratory effects of the siRNAs. Consistent with these results, differential effects of SK1- and SK2-selective siRNAs on signaling proteins, including p53, p21, ERK1, ERK2, FAK, and VCAM1, indicate that SK1 and SK2 have only partially overlapping functions in tumor cells. Overall, these data indicate that loss of SK2 has stronger anticancer effects than does suppression of SK1. Consequently, selective inhibitors of SK2 may provide optimal targeting of this pathway in cancer chemotherapy.

Figures

Figure 1
Figure 1. Effects of SK siRNA transfection on the mRNA levels of SK1 and SK2
A) Relative expression of SK mRNAs in 3 cancer cell lines. Data are presented as percentage of SK1 in A498 cells. B) Cells were transfected with siRNAs as indicated, and qPCR was performed after the indicated times to determine the relative expression of SK1 (open bars) and SK2 (filled bars), compared with control (siNC). Data represent the mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus control.
Figure 2
Figure 2. Effects of SK siRNA transfection on the expression of SK1 and SK2 protein
At 72 hr after transfection, A498 cells were probed with antibodies against SK1 (A, top row) or SK2 (A, bottom row) and visualized with a fluorescent secondary antibody. Images were acquired with identical exposure parameters, and are representative of at least 5 areas from each sample. B) Expression above was quantified by imaging pixel intensity/cell, and the relative protein expression levels of SK1 (open bars) and SK2 (filled bars) were calculated relative to control (siNC). C) The enzymatic activity of SK1 was determined relative to control (siNC-treated) cells. Data represent the mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus control.
Figure 3
Figure 3. Effects of SK siRNA transfection on sphingolipid profiles
A498 cells were transfected with siRNAs as indicated, and sphingolipid analyses were performed 72 hr later. Bars indicate the ratio of the lipid mass relative to control (siNC). Data are mean ± SEM of three independent experiments. *p<0.05, **p<0.01 versus control.
Figure 4
Figure 4. Effects of SK siRNA transfection on the cell cycle profile and proliferation
A498 (A, D), Caki-1 (B, E) or MDA-MB-231 (C, F) cells were harvested 72 hr after siRNA transfection (A, B, C) for cell cycle distribution determination, or cell-number-quantified to plot growth curves (D, E, F). For A498 cells (A, D), 1 μM S1P was added to certain samples as indicated. Data (A, B and C) are mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus siNC control. Data (D, E and F) are mean ± SD and representative of at least two independent experiments.
Figure 5
Figure 5. Effects of SK siRNA transfection on signaling proteins
A498 cells were transfected with siRNA as indicated for 72 hr. A) Cells were harvested and immunoblotting was conducted with the indicated antibodies and quantified by densitometry. The expression of the indicated proteins is normalized to beta-actin. B) qPCR was performed to determine the expression of mRNA for ERK1 (open bars) or ERK2 (filled bars) relative to control (siNC) after normalization to GAPDH. C) Cell cycle histogram of siERK1 and siSK co-transfected cells. Data are mean ± SEM of three independent experiments, except that C represents a single experiment. *p<0.05, **p<0.01, ***p<0.001 versus control.
Figure 6
Figure 6. Effects of SK siRNA transfection on cell migration
A498 (A, C and D) or MDA-MB-231 (B) cells were transfected with siRNAs as indicated and transwell migration and gel invasion assays were performed. In the experiment depicted in panel C, 1 μM S1P was added to both sides of the migration chambers. A) Photos of cells having passed through uncoated (migration) or coated (invasion) filters are representative of at least 5 fields per treatment. Bars in A, B and C represent the percentage of the migratory (open bars) of invasive (filled bars) cells compared with control (siNC). D) Immunoblotting was conducted with the indicated antibodies and quantified. Data are mean ± SEM of three independent experiments except C and D (mean ± SD of triplicates). *p<0.05, **p<0.01, ***p<0.001 versus control.

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