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. 2016 May;35(21):2801-12.
doi: 10.1038/onc.2015.330. Epub 2015 Sep 14.

Ceramide Limits phosphatidylinositol-3-kinase C2β-controlled Cell Motility in Ovarian Cancer: Potential of Ceramide as a Metastasis-Suppressor Lipid

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Free PMC article

Ceramide Limits phosphatidylinositol-3-kinase C2β-controlled Cell Motility in Ovarian Cancer: Potential of Ceramide as a Metastasis-Suppressor Lipid

K Kitatani et al. Oncogene. .
Free PMC article

Abstract

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2β (PI3KC2β) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2β-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2β and affect its compartmentalization, thereby suppressing PI3KC2β activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2β knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2β in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2β-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

Figures

Figure 1
Figure 1. Effects of C6-ceramide on motility of ovarian cancer cells
(A and B) SKOV3 cells, growing in DMEM supplemented with 10% FBS, were incubated with vehicle (control) or 100 nM wortmannin for 30 min. After treatment, cells were fixed followed by staining with phospho-Akt (green), TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Imaging was performed by confocal microscopy, and representative images are shown (A). Formation of lamellipodia was assessed as described in “Materials and Methods” (B). The data shown (mean ± SEM, n = 3) are the percentage of cells forming lamellipodia. (C and D) SKOV3 cells were transfected with 20 nM siRNAs for control-1 (n = 12), control-2 (n = 8), PIK3CA (n = 6), PIK3CB (n = 4), PIK3CG (n = 4), PIK3CD (n = 5), PIK3C2A (n = 4), PIK3C2B (n = 7), PIK3C2G (n = 4), or PIK3C3 (n = 4). After 48 h transfection, cells were fixed followed by staining with TRITC-conjugated phalloidin (white) and Hoechst 33342 (blue). Imaging was performed by confocal microscopy, and representative images are shown (C). Formation of lamellipodia was assessed as described in “Materials and Methods” and yellow allows show lamellipodia (D). Data shown (mean ± SEM, at least four independent experiments) are the percentages relative to cells treated with control-1 siRNAs. Statistical analyses were performed by unpaired, student t-test. *, P < 0.02 and P < 0.001 compared with control-1 and control-2, respectively; **, P < 0.02 and P < 0.002 compared with control-1 and control-2. (E) SKOV3 cells were transfected with the indicated siRNA for 48 h. Extracted proteins were submitted to immunoblot analysis using antibodies specific for PI3KC2β (PIK3C2B) and p110δ (PIK3CD), and β-actin. Equal amounts of protein were loaded in each lane. Results are representative of three independent experiments.
Figure 2
Figure 2. Effects of C6-ceramide on formation of pseudopodia, including lamellipodia and filopodia, in ovarian and breast cancer cells
(A) SKOV3, TOV112D, and MDA-MB-231 cells were treated with or without 10 μM C6-ceramide for 3 h. Cells were fixed followed by staining with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Imaging was performed by confocal microscopy and results are representative of three independent experiments. White allows show lamellipodia. (B and C) SKOV3 cells, grown in DMEM supplemented with 10% FBS, were treated with 10 μM C6-ceramide for the indicated times (B) or with the indicated concentrations of C6-ceramide for 3 h (C). Formation of lamellipodia was assessed as described in “Materials and Methods”. The data shown (mean ± SEM) are the percentages of cells forming lamellipodia. Four or three independent experiments were performed. Statistical analyses were performed by unpaired, student t-test. *, P < 0.05. A, n = 7 for vehicle, n = 8 (0.5 h), 8 (1 h), 13 (3 h), 13 (6 h) for C6-ceramide; B, n = 3.
Figure 3
Figure 3. Identification of ceramide as an inhibitory lipid in cell motility of ovarian cancer cells
SKOV3 cells were simultaneously treated with or without 200 μM fumonisin B1 (FB1) in the absence or presence of 10 μM C6-ceramide for 3 h. (A–D) Lipids were extracted from cells and then sphingolipids (A, long-chain ceramide; B, long-chain hexosylceramide; C, long-chain sphingomyelin; D, sphingosine) were determined by MS. Values are means ± SEM (two independent experiments, n = 4). (E and F) Cells were fixed and stained with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Representative images are shown (E). Formation of lamellipodia was assessed as described in “Materials and Methods” (F). Data shown (mean ± SEM, at least three independent experiments are the percentages of cells forming lamellipodia. n = 3 (Control), 5 (FB1), 4 (C6-Cer), 4 (FB1+C6-Cer). (G) Cell migration was assessed as described in “Materials and Methods”. Data shown (mean ± SEM, four independent experiments, n = 4) are the percentages of vehicle control cells (0.1% ethanol treatment). (H and I) SKOV3 cells were simultaneously treated with 10 μM C6-ceramide in the absence or presence of N-butyldeoxynojirimycin for 3 h. Cells were fixed and stained with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Representative images are shown and white allows show lamellipodia. Formation of lamellipodia was assessed as described in “Materials and Methods”. Data shown (mean ± SEM, three independent experiments, n = 3) are the percentages relative to EtOH. *, P < 0.0001, **, P < 0.002 compared with EtOH. (J) SKOV3 cells were incubated with vehicle (0.1% ethanol), 10 μM C6-ceramide (C6-Cer) or C6-sphingomyelin (C6-SM) for 3 h. Cell migration was assessed as described in “Materials and Methods”. Data shown (mean ± SEM, three independent experiments, n = 3) are percentages of vehicle control cells (0.1% ethanol treatment). Statistical analyses were performed by unpaired, student t-test.
Figure 4
Figure 4. Effects of ceramide on PI3K signaling in ovarian cancer cells
(A) SKOV3 cells were incubated with 10 μM C6-ceramide for 3 h. After treatment, lipids were extracted and phosphatidylinositol 3-phosphate mass was determined by ELISA. Results represent mean ± SEM (two independent experiments, n = 4). (B) SKOV3 cells were treated with the indicated concentration of C6-ceramide for 3 h. Extracted cellular proteins were submitted to immunoblot analysis using antibodies specific for phospho-Akt at Ser473 or Thr308, Akt, phosphor-ERK1/2, and ERK1/2. Equal amounts of protein were loaded in each lane. Results are representative of three independent experiments. (C) SKOV3 cells were simultaneously treated with or without 200 μM fumonisin B1 (FB1) in the absence or presence of 10 μM C6-ceramide for 3 h. Whole-cell lysates were prepared and subjected to immunoblot analysis with antibodies specific for phospho-Akt and Akt. Equal amounts of protein were loaded in each lane. Results are representative of four independent experiments. (D and E) SKOV3 cells were incubated with or without 10 μM C6-ceramide for 3 h followed by treatment with or without 100 ng/ml EGF for 5 min. Cells were fixed and stained with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Representative images are shown (D). Formation of lamellipodia was assessed as described in “Materials and Methods” (E). Data shown (mean ± SEM, three independent experiments, n = 3) are the percentages of cells forming lamellipodia. (F) SKOV3 cells were incubated with or without 10 μM C6-ceramide for 3 h followed by treatment with 100 ng/ml EGF for 2 or 5 min. Proteins were subjected to immunoblot analysis with antibodies specific for phospho-Akt, Akt, and phospo-ERK1/2, ERK1/2, phospho-EGF receptor (EGFR), and EGFR. Equal amounts of protein were loaded in each lane. Results are representative of three independent experiments.
Figure 5
Figure 5. Ceramide interaction with PI3KC2β suppresses cell motility
(A) SKOV3 cells were transfected with empty or V5-tagged PI3KC2β expression vectors. Extracted proteins were submitted to immunoblot analysis with antibodies specific to PI3KC2β or V5. (B) SKOV3 cells transfected with empty or V5-tagged PI3KC2β expression vectors were treated with or without 10 μM C6-ceramide for 3 h. After cell fixation, cells were stained with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Formation of lamellipodia was assessed as described in “Materials and Methods”. Data shown (mean ± SEM, eight independent experiments, n = 8) are the percentages of cells forming lamellipodia. Statistical analyses were performed by unpaired, student t-test. (C) SKOV3 cells transfected with full-length PI3KC2β were treated with vehicle (ethanol) or 30 μM C6-ceramide for 3 h. After cell fixation, cells were stained with Hoechst 33342 (blue) and antibodies specific for V5 antibody (green) and phospho-Akt (Ser473) (red). Arrows show lamellipodia. Results are representative of two independent experiments. (D) Cell supernatants from SKOV3 cells were treated with DMSO, 10 μM biotin, or 10 μM biotinylated C6-ceramide for 1 h at room temperature. Ceramide-interacting proteins were pulled down and submitted to immunoblot analysis using antibodies specific for PI3KC2β, PI3KCα (p110α), and PKCα. Results are representative of three independent experiments. (E) Deletion mutant vectors were constructed. Cell supernatants from SKOV3 cells transfected with those vectors were treated with DMSO, 10 μM biotin, or 10 μM biotinylated C6-ceramide for 1 h at room temperature. Ceramide-interacting proteins were pulled down and submitted to immunoblot analysis using V5 antibody. Results are representative of three independent experiments. (F) SKOV3 cells transfected with the indicated vectors were treated with vehicle (ethanol) or 30 μM C6-ceramide for 3 h. After fixation, cells were stained with V5 antibody (green), TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Results are representative of three independent experiments.
Figure 6
Figure 6. Anti-metastatic effects of ceramide liposomes in vitro and in vivo
(A–D) SKOV3 cells were incubated with TLs or C6-Cer TLs for 12 h. Cells were fixed and stained with Alexa-488-conjugated phalloidin (green). Rhodamine (red) shows the uptake of TLs and C6-Cer-TLs. Imaging was performed by confocal microscopy, and representative images are shown (A). Formation of lamellipodia was assessed as described in “Materials and Methods”. Data shown (mean ± SEM, three independent experiments, n = 3) are the percentages of cells forming lamellipodia (B). Proteins were subjected to immunoblot analysis with antibodies specific for phospho-Akt at Ser473 and Akt. Representative blot images are shown (C). Band signals for phospho-Akt at Ser473 and Akt were quantified, and phospho-Akt/Akt values were calculated. Data shown (mean ± SEM, n = 3 for 21 and 69 μg/ml lipids liposomal lipids, n = 1 for 138 μg/ml lipids liposomal lipids,) are the percentages of phospho-Akt/Akt values relative to TL treatment (21 μg/ml liposomal lipids) (D). (E–G) SKOV3 cells were inoculated into nude mice. Mice were treated with PBS (control, n = 11), TLs (15 mg/kg, n = 8), C6-Cer TLs (15 mg/kg, n = 12) every other day. Body weight was measured every other day (E). Four weeks later, mice were sacrificed, and the number of metastatic nodules was determined (F and G). Arrows show metastatic nodules. Statistical analyses were performed by unpaired, student t-test. P-values were shown in Figure.
Figure 7
Figure 7. Effects of ceramides on the metastasis of PI3KC2β-silenced cells
(A) SKOV3 cells were transfected with 5 nM control-2 or PI3KC2β siRNAs for 48 h followed by treatment with vehicle (ethanol) or 30 μM C6-ceramide for 3 h. Cell migration was assessed as described in “Materials and Methods”. Data shown (mean ± SEM, four independent experiments, n = 4) are the percentages of vehicle control cells (0.1% ethanol treatment). Cell migration in control-2 group was compared with other groups: *P < 0.02; **P < 0.0008; ***P < 0.0007. (B) SKOV3 cells were transfected with 5 nM control-2 or PI3KC2β siRNAs for 24 h and then transfection reagents were removed. Cells were cultured for up to 3 days. Extracted proteins were subjected to immunoblot analysis with PI3KC2β antibody. Results are representative of two independent experiments. (C) SKOV3 cells transfected with siRNAs were inoculated into nude mice. The mice were treated with 15 mg/kg TLs (15 mg/kg, n = 5) or C6-Cer TLs (15 mg/kg, n = 5) on the indicated days. Four weeks later after inoculation, mice were sacrificed and the number of metastatic nodules was determined. Metastasis in control-2 group was compared with other groups: *P < 0.02; **P < 0.05; ***P < 0.007. All statistical analyses were performed by unpaired, student t-test.

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