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

Abstract

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.

Keywords: ceramide kinase; metastatic breast cancer cells; migration and invasion.

Figure 1

Ceramide kinase (CerK) mRNA expression and activity in parental and metastatic MDA-MB-231 cells and in MCF-7 cells. (A) Confluent cells, either parental MDA-MB-231 (MDA), the lung metastatic subline 4175, or the bone-metastatic subline 1833, and MCF-7 were taken for RNA extraction and quantitative PCR analysis of CerK and 18S RNA. (B) Confluent cells were incubated for 3 h with 5 μM of C6-NBD-ceramide. Lipids were then extracted, separated by TLC, and analyzed as described in the Methods section. Results are expressed as percentage of parental MDA-MB-231 cells and are means ± SD (n = 4), * p < 0.05, ** p < 0.01, *** p < 0.001 considered statistically significant compared to the parental MDA-MB-231 values.

Figure 2

Effect of the CerK inhibitor NVP-231 on cell migration of parental and metastatic MDA-MB-231 cells. 5 × 10⁴ parental MDA-MB-231 cells (open columns), lung metastatic (4175, light grey columns), and bone metastatic (1833, dark grey columns) cells were seeded onto transwell filters and treated for 20 h with either vehicle (0) or the indicated concentrations of the CerK inhibitor NVP-231 in Dulbecco’s Modified Eagle Medium (DMEM)/1% fetal bovine serum (FBS). Migrated cells were determined as described in the Methods section. Representative pictures are shown in Supplementary Figure S2. Data are expressed as percentage of control parental MDA-MB-231 cells migrated into the lower chamber and are the means ± SD (n = 3). *** p < 0.001 compared to vehicle-treated parental MDA-MB-231 cells; ^### p < 0.001 compared to the vehicle-treated 4175 or 1833 cells.

Figure 3

Effect of the CerK inhibitor NVP-231 on cell invasion of parental and metastatic MDA-MB-231 cells. 5 × 10⁴ parental MDA-MB-231 cells (open columns), lung metastatic (4175, grey columns), and bone metastatic (1833, black columns) cells were seeded onto Matrigel-coated transwell filters and treated for 48 h in the absence (−) or the presence (+) of NVP-231 (1 μM) in DMEM/1% FBS. Invaded cells were determined as described in the Methods section. Representative images are shown in Supplementary Figure S4. Data are expressed as percentage of parental MDA cells and are means ± SD (n = 3). * p < 0.05 compared to vehicle-treated 4175 cells.

Figure 4

Effect of CerK knockdown on cell migration of metastatic MDA-MB-231 cells. (A,B) Lung metastatic (4175) and bone metastatic (1833) MDA-MB-231 cells that were stably transduced with a lentiviral construct containing CerK small hairpin RNA (shRNA) (CerK-kd) or a control construct (ctrl) were analyzed for CerK mRNA expression (A) and CerK activity (B), as described in the Methods section. (C) 5 × 10⁴ control cells (ctrl) or the CerK-kd 4175 and 1833 cells were seeded onto transwell filters and allowed to migrate for 20 h in DMEM containing 1% FBS. (D) 5 × 10⁴ control cells (ctrl) or the CerK-kd 4175 and 1833 cells were seeded onto Matrigel-precoated transwell filters, as described in the Methods section, and incubated for 24 h in growth medium to allow for invasion. Migrated and invaded cells were quantified as described in the Methods section. Representative pictures are shown in Supplementary Figures S5 and S6. Data are expressed as percentage of ctrl cells and are means ± SD (n = 3 for A–C, n = 6 for D). * p < 0.05, *** p < 0.001 compared to 4175 ctrl cells; ^### p < 0.001 compared to 1833 ctrl cells.

Figure 5

Effect of CerK overexpression on cell migration of parental MDA-MB-231 cells and MCF-7 cells. MDA-MB-231 cells (A–C) or MCF-7 cells (D,E) were transiently transfected with either an empty vector (pcDNA3.1) or a vector containing the human CerK cDNA (hCerK). 48 h after transfection, cells were examined in a CerK activity assay (A,D), in a cell migration assay (B,E), or in an invasion assay (C), as described in the Methods section. Representative pictures of invaded cells are shown in Supplementary Figure S7. Data are expressed as percentage of pcDNA3.1 controls and are means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to pcDNA3.1-treated control cells.

Figure 6

Effect of CerK, MEK, phosphoinositide 3-kinase (PI3K), and RhoA-dependent protein kinase (ROCK) inhibition on migration of CerK-overexpressed parental MDA-MB-231 cells. 5 × 10⁴ parental MDA-MB-231 cells transfected with the human CerK cDNA in a pcDNA3.1 vector (CerKoe) cells were seeded onto transwell filters and incubated for 20 h in DMEM/1% FBS in the absence (Co) or the presence of the CerK inhibitor NVP-231 (NVP, 1 μM), the MEK inhibitor U0126 (10 μM), the PI3K inhibitor LY294002 (LY, 10 μM), and the RhoA-dependent protein kinase (ROCK) inhibitor Y27632 (10 μM). Migrated cells were determined as described in the Methods section. Data are expressed as % of CerKoe control cells and are means ± SD (n = 3), ^## p < 0.01, ^### p < 0.001 considered statistically significant compared to the CerKoe control values.

Figure 7

Effect of CerK inhibition, downregulation, and overexpression on phosphorylation of Akt in MDA-MB-231 and MCF-7 cells. (A) Confluent 4175 and 1833 cells were incubated for 24 h with either vehicle (Co) or NVP-231 (NVP, 1 μM). (B) Then, 4175 and 1833 cells transduced with either a control vector (Ctrl) or a CerK shRNA construct (CerK-kd) were incubated for 24 h in serum-free DMEM. (C,D) Parental MDA-MB-231 cells (C) and MCF-7 cells (D) were transiently transfected with an empty vector (pcDNA3.1) or a vector containing the human CerK cDNA (hCerK) for overexpression. Confluent cells were incubated for 20 h in serum-free DMEM. Thereafter, protein lysates were homogenized and separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blotting using antibodies against phospho-Ser⁴⁷³-Akt, total Akt, phospho-ERK1/2, and β-actin. Results show triplicates of one representative experiment from at least three independent determinations. Bands corresponding to phospho-Akt and total Akt were densitometrically evaluated and are depicted in Supplementary Figure S9.