Ceramide Kinase Is Upregulated in Metastatic Breast Cancer Cells and Contributes to Migration and Invasion by Activation of PI 3-Kinase and Akt

Ceramide kinase (CerK) is a lipid kinase that converts the proapoptotic ceramide to ceramide 1-phosphate, which has been proposed to have pro-malignant properties and regulate cell responses such as proliferation, migration, and inflammation. We used the parental human breast cancer cell line MDA-MB-231 and two single cell progenies derived from lung and bone metastasis upon injection of the parental cells into immuno-deficient mice. The lung and the bone metastatic cell lines showed a marked upregulation of CerK mRNA and activity when compared to the parental cell line. The metastatic cells also had increased migratory and invasive activity, which was dose-dependently reduced by the selective CerK inhibitor NVP-231. A similar reduction of migration was seen when CerK was stably downregulated with small hairpin RNA (shRNA). Conversely, overexpression of CerK in parental MDA-MB-231 cells enhanced migration, and this effect was also observed in the non-metastatic cell line MCF7 upon CerK overexpression. On the molecular level, CerK overexpression increased the activation of protein kinase Akt. The increased migration of CerK overexpressing cells was mitigated by the CerK inhibitor NVP-231, by inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway and the Rho kinase, but not by inhibition of the classical extracellular signal-regulated kinase (ERK) pathway. Altogether, our data demonstrate for the first time that CerK promotes migration and invasion of metastatic breast cancer cells and that targeting of CerK has potential to counteract metastasis in breast cancer.


Suppl Fig. S2
Representative light microscopy images of the effect of the inhibitor NVP-231 on migration of parental and metastatic MDA-MB-231 cells.
5 × 10 4 parental MDA-MB-231 cells, lung metastatic (4175) and bone metastatic (1833) cells were seeded onto transwell filters and treated for 20 h with either vehicle (0 nM) or the indicated concentrations (in nM) of the inhibitor NVP-231. Light microscopic pictures were taken of cells, that migrated through the transwell membrane into the lower chamber (10x magnification, Zeiss Observer Z1).

cells.
After treatment with 1 µM of NVP-231 for 24 h, cells were incubated with 5 µM of C6-NBD-ceramide for 3 h. Lipids were then extracted, separated by TLC and analysed as described in the Methods section. Results are expressed as % of parental MDA-MB-231 cells and are means ± SD (n=3), * p< 0.05, ** p< 0.01 considered statistically significant compared to the ctrl values.

Representative fluorescent images of the effect of the inhibitor NVP-231 on invasion of parental and metastatic MDA-MB-231 cells.
Cells were seeded at a density of 2,5 x 10 5 onto Matrigel-precoated transwell filters as described in the Methods section, and incubated for 48 h with either vehicle (0 nM) or 1000 nM of NVP-231 in growth medium to allow invasion. Thereafter, the transwell filters were removed and invaded cells were stained for 15 min with DAPI (1 µg/ml in methanol), and quantified in five random fields for one sample under a fluorescent microscope (Zeiss Observer Z1).

Suppl Fig S5
Representative light microscopy images of the effect of CerK-kd on migration of metastatic 4175 and 1833 cells.
5 × 10 4 control cells (ctrl) or CerK-kd 4175 and 1833 cells were seeded onto transwell filters and allowed to migrate for 20 h in DMEM containing 1 % FBS. Light microscopic pictures were taken of cells, that migrated through the transwell membrane into the lower chamber (10x magnification, Zeiss Observer Z1).

Representative fluorescent images of the effect of CerK-kd on invasion of metastatic 4175 and 1833 cells.
5 x 10 4 of cells were seeded onto Matrigel-precoated transwell filters as described in the Methods section, and incubated for 24 h in growth medium to allow invasion. Thereafter, the transwell filters were removed and invaded cells were stained for 15 min with DAPI (1 µg/ml in methanol), and quantified in five random fields for one sample under a fluorescent microscope (Zeiss Observer Z1).

Representative fluorescent images of the effect of hCerK overexpression on invasion of parental MDA-MB-231 cells.
5 x 10 4 of cells were seeded onto Matrigel-precoated transwell filters as described in the Methods section, and incubated for 24 h in growth medium to allow invasion. Thereafter, the transwell filters were removed and invaded cells were stained for 15 min with DAPI (1 µg/ml in methanol), and quantified in five random fields for one sample under a fluorescent microscope (Zeiss Observer Z1).