Ceramide Glycosylation by Glucosylceramide Synthase Selectively Maintains the Properties of Breast Cancer Stem Cells

Background: Glucosylceramide synthase catalyzes ceramide glycosylation that regulates the synthesis of glycosphingolipids.

Results: Increased globo-series glycosphingolipids in breast cancer stem cells activate c-Src signaling and β-catenin-mediated transcription up-regulating stem cell factors.

Conclusion: Ceramide glycosylation maintains the stemness of cancer stem cells.

Significance: Glycosphingolipids in cell membrane actively participate in maintaining cancer stem cells.

Abstract

Cancer stem cells are distinguished from normal adult stem cells by their stemness without tissue homeostasis control. Glycosphingolipids (GSLs), particularly globo-series GSLs, are important markers of undifferentiated embryonic stem cells, but little is known about whether or not ceramide glycosylation, which controls glycosphingolipid synthesis, plays a role in modulating stem cells. Here, we report that ceramide glycosylation catalyzed by glucosylceramide synthase, which is enhanced in breast cancer stem cells (BCSCs) but not in normal mammary epithelial stem cells, maintains tumorous pluripotency of BCSCs. Enhanced ceramide glycosylation and globotriosylceramide (Gb3) correlate well with the numbers of BCSCs in breast cancer cell lines. In BCSCs sorted with CD44⁺/ESA⁺/CD24⁻ markers, Gb3 activates c-Src/β-catenin signaling and up-regulates the expression of FGF-2, CD44, and Oct-4 enriching tumorigenesis. Conversely, silencing glucosylceramide synthase expression disrupts Gb3 synthesis and selectively kills BCSCs through deactivation of c-Src/β-catenin signaling. These findings highlight the unexploited role of ceramide glycosylation in selectively maintaining the tumorous pluripotency of cancer stem cells. It speculates that disruption of ceramide glycosylation or globo-series GSL is a useful approach to specifically target BCSCs specifically.

Introduction

Cancer stem cells (CSCs),³ which have been characterized with surface markers in tumors, possess the malignant capacities of self-renewal and pluripotency, thus initiating tumorigenesis and driving tumor progression (1–4). In human breast cancer, the CD44⁺/ESA⁺/CD24^−/low cells have been tested as breast cancer stem cells (BCSCs), because they are able to differentiate into cells with diverse phenotypes and have tumorous pluripotency to generate mammary tumors and metastases in vivo (3, 5). BCSCs, like other CSCs, give rise to tumor resistance to chemotherapy and radiation (6–8). As a cause of tumor metastasis and recurrence, CSC is an adverse prognostic factor for cancer patients (9–11) and a critical target to eradicate cancers (12–15). CSCs are distinguished by loss of tissue homeostasis control and often display unlimited proliferation and growth from normal stem cells, even though both share the similar properties in self-renewal, pluripotency, and resistance to cytotoxins (2, 16). Targeting CSCs pharmacologically for therapeutic purpose requires understanding by which mechanisms CSCs maintain their tumor behaviors.

Several cellular signals including Wnt, Notch, and Hedgehog have been reported to be implicated in the self-renewal ability and pluripotency of normal stem cells in mammary gland development or remodeling and of BCSCs in cancer pathogenesis (5, 16–19). It is not clear how BCSCs, like other CSCs, maintain tumor pluripotency without tissue homeostasis control. Glycosphingolipids (GSLs), particular globo-series GSLs, are common markers of undifferentiated embryonic stem cells (ESCs) (20–22). As essential components of lipid rafts, particularly GSL-enriched microdomains in plasma membrane, GSLs actively mediate cellular signals, gene regulation, and functions (23–26). GSLs may play a vital role in maintaining ESCs, because deletion of GSLs induces apoptosis of ESCs and stops embryo development in ugcg (encoding glucosylceramide synthase, GCS) knock-out mouse (27). GCS catalyzes ceramide glycosylation and is a limiting enzyme controlling the synthesis of GSLs (28, 29). Enhanced ceramide glycosylation by GCS converts ceramide to GSLs, conferring multidrug resistance of cancer cells (30, 31). It has been reported that GCS is overexpressed in metastatic breast tumors (32, 33) and inhibition of GCS decreases lung tumor metastasis (34). These studies suggest that ceramide glycosylation by GCS is associated with CSC behaviors. We examined the correlation of ceramide glycosylation with BCSCs in drug resistance and tumorigenesis.

EXPERIMENTAL PROCEDURES

Cell Culture and Treatments

Human MCF-7 breast cancer cells and the doxorubicin-selected subline MCF-7/Dox were kindly provided by Dr. Kapil Mehta (M. D. Anderson Cancer Center, Houston, TX). MCF-7/Dox cells were derived from MCF-7 cells by stepwise culture with doxorubicin (35). Human MCF-12A mammary epithelial cells were purchased from American Type Culture Collection (Manassas, VA). The MCF-7 and MCF-7/Dox were cultured in RPMI 1640 medium containing 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 584 mg/liter l-glutamine. MCF-12A cells were cultured in Dulbecco's modified Eagle's medium/F-12 (1:1) with 5% horse serum, insulin (5 μg/ml), hydrocortisone (500 ng/ml), human epidermal growth factor (20 ng/ml), and cholera toxin (100 ng/ml). All of the cells were maintained in an incubator humidified with 95% air and 5% CO₂ at 37 °C. Cell lines were authenticated in November 2010 at the John Hopkins University Fragment Analysis Facility (Baltimore, MD) by profiling short tandem repeats for breast cells. FBS was purchased from HyClone (Logan, UT), medium was from Invitrogen, and other reagents from Sigma-Aldrich.

A mixed backbone oligonucleotide (MBO) was designed to target the ORF 18–37 of human GCS and designated as MBO-asGCS (36, 37). For MBO-asGCS treatment, cells (2 × 10⁶ cells/100-mm dish) were grown in 10% FBS RPMI 1640 medium overnight, and MBO-asGCS (100 nm) were introduced into cells using Lipofectamine 2000 (Invitrogen). The cells were cultured with MBO-asGCS in 10% FBS RPMI 1640 medium for 7 days. An additional transfection of MBO-asGCS under the same conditions was conducted on day 4. MBO-asGCS was synthesized and purified by reverse phase HPLC and desalting in Integrated DNA Technologies (Coralville, IA).

Cell Viability Assay

Cell viability was analyzed by quantification of ATP, an indicator of metabolically active cells using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI), as described previously (38). Briefly, cells (4,000 cells/well) were grown in 96-well plates with 10% FBS RPMI 1640 medium for 24 h. MBO-asGCS was introduced into cells by Lipofectamine 2000 (vehicle control) in Opti-MEM reduced serum medium for 4 h of incubation. The cells were then incubated with increasing concentrations of doxorubicin in 5% FBS medium for another 72 h. Cell viability was determined by the measurement of luminescent ATP in a Synergy HT microplate reader (BioTek, Winooski, VT), following incubation with CellTiter-Glo reagent (Promega).

Colony Formation Assay

The cells (10,000 cells/800 μl) were suspended in 10% FBS RPMI 1640 medium containing 0.2% agarose and overlaid onto 24-well plates containing a solidified bottom layer of 0.3% agarose (600 μl) in 10% FBS RPMI 1640 medium. Once the top layer solidified, 600 μl of 10% FBS RPMI 1640 medium was placed on the top to keep the wells moist. For MBO-asGCS treatment, the cells were pretreated with MBO-asGCS as described above. In addition, the MBO-asGCS (200 nm) was added with the 0.2% agarose medium (top layer). The plates were incubated for 1 week until the colonies were visible. The cell numbers of colonies were determined by the measurement of luminescent ATP in a Synergy HT microplate reader, following incubation with CellTiter-Glo reagent for 30 min, at room temperature with gentle shaking.

Tumor Sphere Assay

It was performed as described previously with minor modification (39, 40). Briefly, after sorting, BCSCs or CD44⁻/CD24⁻ cells (5,000 cells/well) were plated in ultralow attachment 24-well plates (Corning, Lowell, MA) with DMEM/F-12 (1:1) medium containing insulin (5 μg/ml), human basic fibroblast growth factor (10 ng/ml), human epidermal growth factor (20 ng/ml), and 0.4% BSA. The BCSCs were treated with MBO-asGCS (100 nm) for 7 days, and the medium was refreshed on day 4. The tumor spheres were observed and photomicrographed.

Cell Sorting

Sorting was performed using magnetic beads (Dynabeads) as described in the manufacturer's protocol. Briefly, after harvest with trypsin-EDTA and PBS pH 7.4 wash, cells (1 × 10⁷ cells) were incubated with mouse anti-human CD24 monoclonal antibody (25 μl; Santa Cruz Biotechnology, Santa Cruz, CA) in 1 ml of cells sorting buffer (PBS containing 0.1% BSA and 2 mm EDTA, pH 7.4) at 4 °C for 10 min. After removal of unbound antibody by centrifugation and PBS washing, the cells were incubated with goat anti-mouse IgG antibody-conjugated Dynabeads M-280 (1 × 10⁷ beads/ml; Invitrogen) at 4 °C for 30 min and placed in a MagCellect magnet (R & D Systems, Minneapolis, MN) for 1 min. An unbound fraction of cells (CD24⁻) was collected for flow cytometry analysis and immunohistochemistry. To separate BCSC and other subpopulations, the CD24⁻ fraction from MCF-7/Dox cells (1 × 10⁷ cells) was further incubated with 25 μl of mouse anti-human CD44 monoclonal antibody (Sigma) at 4 °C for 10 min and with goat anti-mouse IgG antibody-conjugated Dynabeads M-280 (1 × 10⁷ beads/ml) for 20 min following removal of unbound antibody. The CD44⁻ fraction (CD44⁻/CD24⁻) were used for further analysis and CD44⁺ fraction (CD44⁺/CD24⁻) was cultured in 10% FBS RPMI 1640 medium for 48 h to dissociate antibody Dynabeads. After incubation with mouse anti-ESA/flotillin-2 antibody (25 μl/10 × 10⁷ cells; from Santa Cruz) and then anti-mouse IgG antibody-conjugated Dynabeads M-280 (1 × 10⁷ beads/ml), the ESA⁺ fraction (ESA⁺/CD44⁺/CD24⁻) and ESA⁻ fraction (ESA⁻/CD44⁺/CD24⁻) were harvested after magnetic separation and cultured for further experiments. The MBO-asGCS (100 nm) treatment of MCF-7/Dox cells was performed as described above.

Flow Cytometry

Flow cytometry was performed as described previously (15). For analysis of BCSCs, the CD24⁻ cells sorted from each cell line were incubated with Alexa Fluor® 488 anti-CD44 mouse antibody (5 μl/10⁶ cells; BioLegend, San Diego, CA) and Alexa Fluor® 647 anti-ESA antibody (5 μl/10⁶ cells; BioLegend) in blocking buffer (PBS containing 5% serum) at 4 °C for 30 min. After washing off unbound antibodies, the cells were resuspended in 1 ml of PBS and immediately analyzed on a BD FACSCalibur with the BD CellQuest Pro program (BD Bioscience, San Jose, CA) and FlowJo v10 (Tree Star, Ashland, OR). To identify CD44⁺/ESA⁺ cells, each sample was incubated in RPMI medium containing serum to determine the autofluorescence, as negative control. To analyze BCSCs in tumors, cell suspensions were prepared immediately after resection (<10 min). Tumor tissues (∼60 mg) were cut into small pieces (<1 mm) and incubated with collagenase IV (500 units/ml; purchased from Sigma) in RPMI 1640 and incubated at 37 °C for 2 h with shaking (100 rpm). Further, the cells were passed through a 70-μm cell strainer and washed twice with PBS.

Cellular Ceramide Glycosylation and GSL Analysis

Ceramide glycosylation that is catalyzed in cells was analyzed as described previously (41). Cells were grown 24 h in 35-mm dishes (5 × 10⁶ cells/dish) in 10% FBS RPMI 1640 medium and MBO-asGCS (100 nm) was introduced as described above. After 12 h of growth in 10% FBS RPMI 1640 medium, the cells were switched to 1% BSA RPMI 1640 medium containing 5 μm NBD C₆-ceramide complexed to BSA (Invitrogen). After 2 h of incubation at 37 °C, the lipids were extracted and resolved on Partisil high performance thin layer chromatography (HPTLC) plates with fluorescent indicator (number 4806-410; Whatman, Florham Park, NJ) in a solvent system containing chloroform, methanol, and 3.5 n ammonium hydroxide (85:15:1, v/v/v) as described previously (41). NBD C₆-glucosylceramide and NBD C₆-ceramide were identified using AlphaImager HP imaging system (Alpha Innotech, San Leandro, CA) and quantitated on a Synergy HT multi-detection microplate reader. For quantification, calibration curves were established after TLC separation of NBD C₆-ceramide (Invitrogen) and NBD C₆-glucosylceramide (N-hexanol-NBD-glucosylceramide; Matreya, Pleasant Gap, PA).

The endogenous GSL were extracted and analyzed as described previously (25, 42). Briefly, cellular lipids were extracted with chloroform/methanol/water (1:1:1, v/v/v) from each cell line or subpopulation after vehicle or MBO-asGCS treatments. Extracted lipids were resuspended in chloroform/methanol (1:1, v/v) and applied to Partisil HPTLC plates with fluorescent indicator. Lipids were resolved using the solvent systems of chloroform/methanol/water (65:35:8, v/v/v) for GSLs. HPTLC plates were dipped for 10 s in 0.02% primuline (w/v; purchased from Sigma) in acetone/water (4:1, v/v). Fluorescence TLC profile graph was visualized under long wave UV light (360 nm) and captured using AlphaImager HP system (Alpha Innotech). Neutral GSL Qualimix (Matreya) were used as TLC standards. Globotriosylceramide (Gb3) of each sample was quantified by fluorescence intensity against the standard curve established using Gb3 lipids (Matreya) and normalized against cellular proteins.

MALDI-MS and MS Analysis of GSLs

MALDI-MS profiling of permethylated GSLs was performed as described previously (43). Briefly, the extracted GSLs (from 7.5 × 10⁵ cells) were incubated with leech ceramide glycanase (50 milliunits/100 μl) (44) in 50 mm sodium acetate, pH 5.5, containing 0.1% sodium cholate at 37 °C for 16 h. The released glycans were separated from the ceramides and detergent by passing through a Sep-Pak C18 cartridge (Waters, Milford, MA), and further desalted by a porous graphitized carbon solid phase extraction cartridge. Permethylation was carried out by the NaOH/Me₂SO slurry method as described previously (45). MALDI-MS analysis of the derivatives were carried out in positive ion mode on a ABI 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA) using 2′,4′,6′-trihydroxyacetophenone as the MALDI matrix. Gb3 mass intensity was normalized against cellular proteins of each sample.

Western Blot Analysis

After treatment, cells or tissue homogenates were lysed using Nonidet P-40 cell lysis buffer (BIOSOURCE, Camarillo, CA). The amount of total proteins was measured using a BCA protein assay kit (Pierce). Equal amounts of detergent-soluble proteins (50 μg/lane) were resolved using 4–20% gradient SDS-PAGE (Invitrogen). After transferring, the blots were blocked in 5% fat-free milk in PBS for 1 h at 26 °C. The blots were then incubated with specific primary antibodies against GCS, FGF-2, Oct-4, CD44, c-Src, phosphorylated c-Src, β-catenin, and phosphorylated β-catenin (1:200 to 1:5000 dilution) overnight at 4 °C and then with respective horseradish peroxide-conjugated secondary antibodies (1:2500 dilution) for 1 h at 26 °C after washing. The proteins were detected using enzyme-linked SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL) as described previously (25, 30). Endogenous GAPDH was used as a loading control for each sample.

Tumor-bearing Mice and Treatments

All of the animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Louisiana at Monroe and were handled in strict accordance with good animal practice as defined by National Institutes of Health guidelines. Athymic nude mice (Foxn1^nu/Foxn1⁺, 4–5 weeks, female) were purchased from Harlan (Indianapolis, IN) and maintained in the vivarium at University of Louisiana at Monroe. Animal studies were conducted as described previously (25, 36). Briefly, cells of each subpopulation (BCSC, CD44⁻/CD24⁻) were cultured 24 h after sorting and resuspended in serum-free RPMI 1640 medium (20 μl). After anesthesia, cell suspensions (2.5 × 10⁴ cells/20 μl or 1 × 10⁵ cells/20 μl) were inoculated into the second mammary gland fat pad just beneath the nipple of each mouse implanted with 17β-estradiol tablets (0.72 mg, 90 days release; Innovative Research of America, Sarasota, FL) to generate orthotopic breast tumors. The mice were monitored by measuring tumor volume, body weight, and clinical observation. Tumor volume (V) was calculated by V = L/2 × W², where L was the length, and W was the width of tumors. Tumors and metastasis of mice were examined by a pathologist with hematoxylin and eosin stain of tissues sections at Louisiana State University Health Science Center (Shreveport, LA).

Statistical Analysis

All of the experiments were conducted in triplicate and repeated two times. The data were analyzed by using Prism version 4 (GraphPad software, San Diego, CA) or SAS 9.2 (SAS Institute, Gary, NC) and presented as the means ± S.D. Two-tailed Student's t tests were used to compare the continuous variables between groups, and Fisher's extract test was used to compare the proportion between groups. All p < 0.05 was considered statistically significant.

RESULTS

GCS Is Associated with BCSCs in Drug Resistance and Tumorigenicity of Cancer Cells

Different cancer cell lines display their own behavior in response to anticancer drugs and in tumor formation. In assessment of cell response to doxorubicin, we found that the half-maximal inhibitory concentration (IC₅₀) value for doxorubicin in MCF-7/Dox cells was 30-fold (9.8 μm versus 0.33 μm; p < 0.001) higher than the MCF-7 breast adenocarcinoma cells or human MCF-12A mammary epithelial cells (Fig. 1A). These results are consistent with previous reports, because MCF-7/Dox cells, which have been selected with doxorubicin culture from MCF-7 cells, are resistant to doxorubicin (35). Furthermore, MBO-asGCS treatment (100 nm) to silence GCS expression (36) in the present study significantly decreased the IC₅₀ for doxorubicin by 13-fold (9.8 μm versus 0.75 μm; p < 0.001) in MCF-7/Dox cells (Fig. 1A). Examining colony formation in soft agar, we found that MCF-7/Dox cells formed 160% more colonies (9,325 cells versus 5,715 cells; p < 0.001) than MCF-7 cells or MCF-12A cells (Fig. 1B); in contrast, MBO-asGCS treatment reduced the colonies by 2-fold (9,315 cells versus 4,702 cells; p < 0.001) in MCF-7/Dox cells (Fig. 1B). These results show that GCS expression is associated with drug resistance and tumorigenicity, which are characteristics of CSCs in these cancer cell lines.

FIGURE 1.

Cell response to doxorubicin and colony formation of human breast cancer cells. A, cell response to doxorubicin. The cells were exposed to increasing concentrations of doxorubicin (0–20 μm) in 5% FBS medium for 72 h. The half-maximal inhibitory concentration (IC₅₀) for cell viability was calculated using Prism software after measurement. Dox/asGCS, MCF-7/Dox cells treated with MBO-asGCS (100 nm). *, p < 0.001compared with MCF-7 cells; **, p < 0.001 compared with vehicle control of MCF-7/Dox cells. B, colony formation in soft agar. The colonies (cell numbers) formed in agarose (0.2%, 7 days) were measured using CellTiter-Glo reagent.

The cells with CD44⁺/ESA⁺/CD24⁻ phenotype have been characterized as BCSCs in tumors and in cancer cell lines (3, 6, 46). To test whether BCSC is a cause of drug resistance and tumorigenicity presenting in MCF-7/Dox cells, we analyzed BCSCs with CD44⁺/ESA⁺/CD24⁻ phenotype in these cell lines. In CD24 separation, ∼36% of MCF-7 and 54% of MCF-7/Dox were CD24⁻ cells. Different from these cancer cell lines, only 28% of MCF-12A cells were CD24⁻. It was found that the number of BCSCs (CD44⁺/ESA⁺/CD24⁻) in MCF-7/Dox cells was 6-fold (3.1% versus 0.5% of cancer cells; p < 0.001) more than MCF-7 and 10-fold (3.1% versus 0.3% of cells; p < 0.001) more than MCF-12A cells (Fig. 2). Silencing of GCS using MBO-asGCS significantly reduced the BCSCs to 42% (1.3% versus 3.1% of cancer cells; p < 0.001) in MCF-7/Dox cells (Fig. 2).

FIGURE 2.

Breast cancer stem cells in human breast cancer cell lines. A, flow cytometry histograms of CD24⁻ cells. The CD44⁺/ESA⁺ cells (BCSCs) enclosed in ellipses (upper right) were compared with negative controls (CD44⁻, ESA⁻) of the CD24⁻ fraction following magnetic separation of each cell line. B, BCSCs. After magnetic separation (CD24) and flow cytometry analysis (CD44/ESA), BCSCs were determined and presented as the percentages of total cells. Dox/asGCS, MCF-7/Dox cells treated with 100 nm MBO-asGCS for 7 days. *, p < 0.001 compared with MCF-7 cells; **, p < 0.001 compared with vehicle control of MCF-7/Dox cells.

Ceramide Glycosylation Catalyzed by GCS Is Crucial for Tumor Formation

To determine whether ceramide glycosylation plays a role in modulating BCSCs, we examined the correlation of GCS expression, ceramide glycosylation, and endogenous GSLs with BCSCs. Western blotting showed that GCS levels in MCF-7/Dox cells were three times higher than MCF-7 or MCF-12A cells; silencing of GCS using MBO-asGCS substantially reduced GCS to 40% in MCF-7/Dox cells (Fig. 3A). Interestingly, the expression of FGF-2 and Oct-4, which are required for the proliferation of normal stem cells and CSCs (2, 47, 48), was substantially increased with levels of GCS protein in these cells (Fig. 3A). Consistent with protein level, GCS activity in MCF-7/Dox cells was 2-fold (26 pmol/mg versus 13 pmol/mg) higher than MCF-7 cells or MCF-12A cells (Fig. 3B). The GSL profiling analyzed by HPTLC indicated that GSLs, particularly of Gb3 in MCF-7/Dox cells were 2-fold (32.6 versus 15.4 ng/mg; p < 0.001) greater than MCF-7 and 4-fold (32.6 versus 8.2 ng/mg; p < 0.001) greater than MCF-12A cells (Fig. 3C). Silencing of GCS with MBO-asGCS decreased GSLs, and the Gb3 was reduced to 28% (9.1 ng versus 32.6 ng/mg; p < 0.001) in MCF-7/Dox cells (Fig. 3C). To verify these findings, GSLs further profiled using MALDI-MS and MS/MS analysis (43). The Gb3 levels in MCF-/Dox cells was 250% higher than in MCF-7 cells (10,179 versus 4,139 in absolute height; p < 0.001) and 347% higher than in MCF-12A cells (10,179 versus 2,833 in absolute height; p < 0.001) (Fig. 3, D–F). MBO-asGCS treatment reduced the Gb3 to 33% (3349 versus 10179 in absolute height; p < 0.001) in MCF-7/Dox cells (Fig. 3D). We further examined whether or not GCS itself is able to induce BCSCs in MCF-7 cells. As shown in supplemental Fig. S1, GCS transfection did not significantly enhance either BCSC number or protein levels of FGF-2 and Oct-4 in MCF-7/GCS cells, although these cells overexpressed GCS. These results indicate that GCS and Gb3 modulate the maintenance of BCSCs, possibly through stem cell mediators like FGF-2 and Oct-4.

FIGURE 3.

GCS expression and GSLs in human breast cancer cell lines. Dox/asGCS, MCF-7/Dox cells treated with MBO-asGCS (100 nm) for 7 days. A, Western blot. Detergent-soluble proteins (50 μg/lane) were resolved by 4–20% SDS-PAGE and immunoblotted with anti-GCS, FGF-2, and Oct-4 antibodies, respectively. B, fluorescence chromatogram of GCS activity. The cells were incubated with NBD C₆-ceramide (50 μm, 2 h) in 1% BSA RPMI 1640 medium to measure cellular GCS activity. An equal amount of lipids (5,000 FI) was spotted on an HPTLC plate. Cer, NBD C₆-ceramide; GlcCer, NBD C₆-glucosylceramide synthase. GCS activity is presented as GlcCer generated per cellular proteins (pmol/mg). *, p < 0.001, compared with MCF-7 or MCF-12A cells; **, p < 0.001, compared with vehicle control of MCF-7/Dox cells. C, GSL profiling. Equal amounts of cellular lipids (equal to 100 μg of proteins) were separated by HPTLC and detected by staining with 0.02% primuline and followed by fluorescence imaging. Std, standards of neutral glycosphingolipid Qualimix; GlcCer, glucosylceramide; LC, lactosylceramide. Endogenous Gb3 was quantitatively measured by comparison of the fluorescence intensity to a standard curve and normalized against proteins. *, p < 0.001, compared with MCF-7 or MCF-12A cells; **, p < 0.001, compared with vehicle control of MCF-7/Dox cells. D, Gb3 MS intensities of cancer cells. GSLs were analyzed by using MALDI-MS analysis, and the Gb3 MS intensity was normalized against cellular proteins of each sample. E, MALDI-MS profiles of permethylated GSLs of normal mammary epithelial MCF-12A cells. F, MALDI-MS profiles of permethylated GSLs of MCF-7/Dox breast cancer cells. Lc, lactosylceramide; *, unidentified peaks; Gb3 abs int., absolute MS intensity of Gb3, as calculated by the percentage of Gb3 peak × MS response (the count on the right y axis).

Furthermore, we observed tumor formation of MCF-12A, MCF-7, and MCF-7/Dox cell lines in vivo. After 35 days inoculation, there were no tumors detected in MCF-12A group (zero of five mice). The tumor volume in MCF-7/Dox was 7-fold (109.4 mm³ versus14.4 mm³; p < 0.001; five of five mice) greater than MCF-7 group (four of five mice). Silencing of GCS with MBO-asGCS (4 mg/kg for 2 weeks) retarded the tumor growth in MCF-7/Dox, because the tumor volume was reduced to 11% (12.4 mm³ versus 109.4 mm³; p < 0.001). Assessment of BCSCs (CD44⁺/ESA⁺/CD24⁻) showed that BCSCs from MCF-7/Dox tumors were 4-fold (4.6% versus 1.1% of tumor cells; p < 0.001) larger than MCF-7 tumors. MBO-asGCS treatment decreased BCSCs to 43% (2.0% versus 4.6% tumor cells; p < 0.001) (Fig. 4B). These results indicate BCSCs that highly express GCS are the cause of tumorigenicity of these cell lines in vivo.

FIGURE 4.

BCSCs in tumors. A, flow cytometry histograms of CD44 and ESA cells from tumors. CD24⁻ cells were separated from tumors from each group and analyzed using flow cytometry. The CD44⁺/ESA⁺ cells (BCSCs) enclosed in the ellipses (upper right) were compared with negative controls (CD44⁻, ESA⁻) of the CD24⁻ fraction. B, tumor BCSCs. After magnetic separation (CD24) and flow cytometry analysis (CD44/ESA), BCSCs are presented as the percentage of total tumor cells. Dox/asGCS, MCF-7/Dox tumors were treated with MBO-asGCS (4 mg/kg, intraperitoneal, 14 days). *, p < 0.001 compared with MCF-7 tumors; **, p < 0.001, compared with vehicle treatment of MCF-7/Dox tumors.

Ceramide Glycosylation Determines Stem Properties of BCSCs in Tumor Formation and Metastasis

To determine whether or not BCSCs rely on ceramide glycosylation, we sorted BCSCs and other subpopulations from MCF-7/Dox cells and examined their tumorigenicity and ceramide glycosylation. Consistent with previous reports (6), it was found that the clonogenicity of BCSCs (CD44⁺/ESA⁺/CD24⁻) was 3-fold greater (19,540 cells versus 5,717 cells; p < 0.001) than the CD44⁻/CD24⁻ subset and significantly higher than other non-stem cell subsets (CD44⁺/CD24⁻/ESA⁻, CD44⁺/CD24⁺, and CD44⁻/CD24⁻) (data not shown here). Conversely, silencing GCS by MBO-asGCS reduced the number of colonies to 45% (8,724 cells versus 19,540 cells; p < 0.001) in BCSCs (Fig. 5A). These results further were confirmed by tumor sphere assay (Fig. 5A, bottom panels). Furthermore, the tumorigenicity of BCSCs and CD44⁻/CD24⁻ cells were examined in athymic nude mice. It was found that BCSCs formed significantly more tumors, and the tumors were bigger than the non-stem cell subset (CD44⁻/CD24⁻) (Table 1 and Fig. 5B). The tumor incident detected in the BCSC group (1 × 10⁵ cells/mouse inoculation; 12 of 12 mice) is statistically significant, 6-fold higher (p < 0.0071) than CD44⁺/CD24⁻ group (one of six mice). Tumors from BCSCs grew aggressively and were 6-fold larger than the non-stem cell subset (1,858 versus 274 mm³; p < 0.001) (Fig. 5B). Moreover, lung metastases were detected in mice injected with BCSCs (two of 12 mice), but not in mice inoculated with CD44⁻/CD24⁻ cells, as indicated in the hematoxylin and eosin staining of lung section (Fig. 5B, bottom panels). However, the difference of metastasis incidence was not significant (p < 0.99) between the two groups, because of small sample size.

FIGURE 5.

Colony formation and tumorigenesis of BCSCs. BCSC and CD44⁻/CD24⁻ subsets were sorted from MCF-7/Dox cells. BCSC/asGCS, BCSCs were treated with MBO-asGCS (100 nm, 7 days). A, colony formation of BCSCs. BCSC and other cell subsets were sorted from MCF-7/Dox cells and plated for colony formation. Bottom panels, tumor spheres were photomicrographed (200 × magnification). B, tumor formation of BCSCs. The cells (1 × 10⁵ cells/mouse) of BCSC (CD44⁺/ESA⁺/CD24⁻) and of the non-stem cell subset (CD44⁻/CD24⁻) were inoculated in the mammary gland of athymic nude mice, and the tumor growth was observed for 84 days. *, only one benign xenograft was found (one of 12) in this group. Bottom panels, hematoxylin and eosin (H&E) staining of lung sections.

TABLE 1.

Breast tumor formation by BCSC and CD44⁻/CD24⁻ cells in athymic nude mice

Cells injected	Tumors formed in mice	Lung metastasis
BCSC
100,000 cells/mouse	12/12a	2/12
25,000 cells/mouse	2/4	0/4
CD44−/CD24−
100,000 cells/mouse	1/4	0/4
25,000 cells/mouse	0/4	0/4

^a The p value of tumor volumes is 0.0071 compared with CD44⁻/CD24⁻ cells.

It has been reported lately that CD44⁺/CD24⁻ subset cells overexpress the mRNA of ugcg and another 15 genes (49). We examined GCS activities in BCSCs (CD44⁺/ESA⁺/CD24⁻) and other subsets in this study. It was found that GCS enzyme activity in BCSCs was 2-fold higher (33 versus 16 pmol/mg; p < 0.001) than in the CD44⁻/CD24⁻ subset (Fig. 6A) and significantly higher than other non-stem cell subsets (CD44⁺/CD24⁻/ESA⁻, CD44⁺/CD24⁺, and CD44⁻/CD24⁻) (data not shown here). Silencing GCS with MBO-asGCS reduced the enzyme activity of BCSCs to 30% (33 versus 10 pmol/mg; p < 0.001) (Fig. 6A). Profiling of GSLs using HPTLC indicated that the levels of GSLs were consistent with the alteration of GCS activities in BCSCs, non-stem cell subset, and BCSCs treated with MBO-asGCS (Fig. 6, A and B). Markedly, Gb3 levels in BCSCs were significantly higher than in non-stem cell subset (CD44⁻/CD24⁻), and MBO-asGCS treatment reduced Gb3 to 37% (46 versus 17 ng/mg; p < 0.001) in BCSCs (Fig. 6B). These results were further confirmed by findings using MALDI-MS and MS/MS analysis. As shown in Fig. 6 (C and D), MBO-asGCS treatment significantly reduced Gb3 level in BCSCs to 50% (15816 versus 31430 in absolute height; p < 0.001). Together, these findings indicate enhanced ceramide glycosylation is a unique characteristic of BCSCs, and it plays a crucial role in maintaining the stemness of BCSCs.

FIGURE 6.

GCS activity and GSLs of BCSCs. BCSC and non-stem cell subset were sorted from MCF-7/Dox cells. BCSC/asGCS, BCSCs were treated with MBO-asGCS (100 nm, 7 days). A, fluorescence chromatogram of GCS activity. Cer, NBD C₆-ceramide; GlcCer, NBD C₆-glucosylceramide; Std, standards of Cer and GlcCer. GCS activity is presented as GlcCer generated per cellular proteins (pmol/mg). *, p < 0.001 compared with non-stem cell subset CD44⁻/CD24⁻; **, p < 0.001, compared with vehicle control of BCSC. *, p < 0.001 compared with non-stem cell subset, CD44⁻/CD24⁻. BCSC/asGCS, BCSCs were treated with MBO-asGCS (100 nm). **, p < 0.001 compared with vehicle control of BCSC. B, GSL profiles. GlcCer, glucosylceramide; LC, lactosylceramide; Std, standards of neutral glycosphingolipid Qualimix. *, p < 0.001 compared with the CD44⁻/CD24⁻ subset; **, p < 0.001 compared with vehicle control of BCSC. C, MALDI-MS profiles of permethylated GSLs of BCSC. D, MALDI-MS profiles of permethylated GSLs of BCSC treated with MBO-asGCS. Lc, lactosylceramide; *, unidentified peaks; Gb3 abs. int., absolute intensity of Gb3.

c-Src/β-Catenin Signaling Modulated by GCS Is Essential for BCSCs

It has been reported that Wnt/β-catenin signaling is critical for the self-renewal of normal mammary epithelial stem cells and BCSCs (17, 18, 50, 51). Globo-series of GSLs can mediate Wnt/β-catenin signaling and cell functions (25, 52, 53). To elucidate how GSLs modulate BCSCs, a stepwise inhibition approach was employed to examine the effects of ceramide glycosylation on BCSC stemness. As shown in Fig. 7, the protein levels of CD44, FGF-2, Oct-4, phosphorylated c-Src, and β-catenin (active form) were substantially increased in BCSCs, as compared with non-stem cells (CD44⁻/CD24⁻); suppression of ceramide glycosylation by MBO-asGCS (100 nm) substantially repressed the protein expression of CD44, FGF-2, and Oct-4, accompanied by a decrease of phosphorylated c-Src and β-catenin (active form) in BCSCs (CD44⁺/ESA⁺/CD24⁻) (Fig. 7, A and B). Consequently, MBO-asGCS decreased BCSCs to 30% in MCF-7/Dox cells (Fig. 7C). FH535 can specifically inhibit β-catenin recruitment of transcription factors and regulation of gene expression (25, 54, 55). We found that FH535 treatments (5 μm) repressed the protein expression of CD44, FGF-2, and Oct-4 by ∼3-fold in BCSCs, and thus decreased BCSCs to 41% in MCF-7/Dox cells (Fig. 7, A and C). It has been reported that pyrazolo pyrimidine (PP2) can inhibit c-Src phosphorylation (25, 56, 57). In this study, PP2 treatments (5 μm) significantly decreased the levels of phosphorylated c-Src and reduced the level of β-catenin to 7% (0.9 versus 13.0 β-catenin/pβ-catenin; p < 0.001) in BCSCs (Fig. 7B). Consistently, PP2 treatment decreased BCSCs to 33% in MCF-7/Dox cells (Fig. 7C). Together, these findings strongly suggest that ceramide glycosylation activates c-Src and β-catenin signaling pathway and mediates the expression of CD44, FGF-2, and Oct-4 to maintain BCSCs.

FIGURE 7.

GCS increases c-Src/β-catenin signaling for BCSC maintenance. The BCSC and CD44⁻/CD24⁻ subsets were sorted from MCF-7/Dox cells. BCSC/asGCS, BCSCs sorted from MCF-7/Dox cells after MBO-asGCS treatment (100 nm for 7 days). A, β-catenin is crucial for GCS up-regulating FGF-2, Oct-4, and CD44 in BCSC. Equal amounts of detergent-soluble proteins (50 μg/lane) were resolved by 4–20% SDS-PAGE and immunoblotted with anti-CD44, FGF-2, Oct-4, and GAPDH antibodies. BCSC+FH535, BCSCs sorted from MCF-7/Dox cells treated with FH535 (β-catenin inhibitor, 5 μm for 24 h). *, p < 0.001 compared with the FGF-2/GAPDH ratio of CD44⁻/CD24⁻ subset; **, p < 0.001 compared with the FGF-2/GAPDH ratio of BCSC. B, c-Src phosphorylation mediates GCS effect on enhancing β-catenin in BCSCs. Equal amounts of detergent-soluble proteins (50 μg/lane) were resolved by 4–20% SDS-PAGE and immunoblotted with anti-GCS, c-Src, p-c-Src, p-β-catenin, β-catenin, and GAPDH antibodies. BCSC+PP2, BCSCs sorted from MCF-7/Dox cells treated with PP2 (c-Src kinase inhibitor, 5 μm for 24 h); p-cSrc, phosphorylated c-Src; pβ-catenin, phosphorylated β-catenin. *, p < 0.001 compared with the β-catenin/GAPDH ratio of CD44⁻/CD24⁻ subset; **, p < 0.001 compared with the β-catenin/GAPDH ratio of BCSCs. C, effects of inhibitions of GCS, c-Src kinases, and β-catenin on BCSCs. BCSCs were analyzed following MCF-7/Dox cells treated with MBO-asGCS (100 nm, 7 days; Dox/asGCS), PP2 (5 μm, 24 h; Dox/PP2), and FH535 (5 μm, 24 h; Dox/FH535). *, p < 0.001 compared with vehicle control of MCF-7/Dox cells.

DISCUSSION

Our results clearly indicate that ceramide glycosylation catalyzed by GCS is important for CSCs in drug resistance and tumorigenesis. BCSCs have been identified with surface markers, such as CD44⁺/ESA⁺/CD24⁻, in established breast cancer cell lines and in tumor specimens (3, 5, 6, 46). In addition to tumorigenesis, the accumulated BCSCs in tumors drive tumor progression to disseminated metastasis and poor response to chemotherapy (8, 10, 58). Among human breast cancer cell lines, the tumorigenesis and drug resistance are reported highly associated with the numbers of BCSCs (5, 6, 49). In the present study, MCF-7/Dox cells that have 3-fold more BCSCs displayed marked resistance to doxorubicin and were aggressive in tumorigenesis (Figs. 1, 2, and 4). Despite GCS overexpression in metastatic breast tumors and causing drug resistance (31, 33), there is no clear experimental evidence linking these to CSCs. Ceramide glycosylation by GCS found in the present study correlated well with BCSCs, as well as their tumorous property in drug resistance and tumorigenicity. MCF-7/Dox cells overexpressed GCS protein and produced 2-fold more Gb3 following enhanced ceramide glycosylation, as compared with MCF-7 breast cancer cells. In contrast, the levels of GCS protein and Gb3, which were consistent with the BCSC numbers, were substantially lower in normal MCF-12A mammary epithelial cells (Fig. 3). Disruption of ceramide glycosylation by silencing of GCS decreased BCSCs in cultured cells and in tumors generated from MCF-7/Dox cells (Figs. 2 and 4); consequently, silencing of GCS reversed drug resistance and eliminated tumorigenesis. These results together indicate that enhanced ceramide glycosylation caused by GCS overexpression is essential for BCSCs retaining drug resistance and tumorigenicity.

With direct evidence, the present study demonstrated that globo-series GSLs play a crucial role in maintaining CSCs. Among GSLs synthesized after ceramide glycosylation, globopentosylceramide (Gb5, Galβ3GalNAcβGalβ4Glcβ1Cer) and monosialyl globopentosylceramide (MSGb5, Siaα2,3Galβ3GalNAcβGalβ4Glcβ1Cer), which are generated from Gb3 and recognized as stage specific embryonic antigen-3 and -4, are broadly used as markers to characterize undifferentiated human ESCs including isolated, derived, and induced pluripotent stem cells (20–22, 60, 61). Gb5 and other globo-series GSLs are synthesized by a serial glycosylation of ceramide catalyzed by GCS, lactosylceramide synthase, Gb3 synthase, and Gb5 synthase, but GCS is the first rate-limiting enzyme for their synthesis in mammalian cells (62, 63). GSLs play important roles in ESCs, because homozygous knock-out of ugcg in mouse prevents the development and differentiation of the gastrulating embryo by massive apoptosis largely in the ectodermal layer (27). Deletion of GSLs by using GCS inhibitor (1-phenyl-2-decanoylamino-3-morpholino-1-propanol), previous study indicates that Gb5 and MSGb5 do not play critical functional roles in maintaining the pluripotency of human ESCs, even though their expression clearly marks undifferentiated ESCs (64). Gb5 and Glob H (Fucα1–2Galβ1–3GalNacβ1–3Galα1–4Galβ1–4Glcβ1) generated from Gb5 by fucosyltransferase are overexpressed on breast cancers and other epithelial cell tumors, such as colon, ovarian, gastric pancreatic, lung, and prostate cancers (28, 65, 66). However, Gb5 and Globo H, which are examined by flow cytometry analysis are expressed with no significant difference between BCSCs (CD45⁻/CD24⁻/CD44⁺) and non-BCSCs (CD45⁻/CD24⁺/CD44⁺ and CD45⁻/CD24⁻/CD44⁻) sorted from tumor specimens of breast cancer patients (66). To clarify the role of GSLs in CSCs, we employed direct approaches to assess GCS protein, ceramide glycosylation, Gb3 by mass spectrometry, BCSC numbers, and their tumorous properties of BCSCs at the present of GCS silencing. Non-stem cell subset (CD44⁻/CD24⁻) presented much lower tumorigenicity and GCS activity than in any other populations (Fig. 5). BCSCs (CD44⁺/ESA⁻/CD24⁻) overexpressed GCS and produced more Gb3. Importantly, disruption of ceramide glycosylation by silencing of GCS inhibited Gb3 in BCSCs (CD44⁺/ESA⁻/CD24⁻), and consequently it eradicated BCSCs, displaying in tumor formation and tumor progression (Figs. 5 and 6).

Ceramide glycosylation by GCS is a unique process by which CSCs maintain their tumorous stemness. Ceramide glycosylation, which is known for reducing cellular ceramide biochemically and protecting cell from ceramide-induced apoptosis, can endow CSC resistance to cytotoxins and anticancer drugs. It has been reported that CSCs (CD55^hi) have high tolerance to ceramide-induced apoptosis, and this resistance is attributed to abundance of sphingomyelin synthase 1 that can converts ceramide to sphingomyelin (67). Because GCS level is higher in BCSCs versus lower in bone marrow stem cells, doxorubicin can enhance BCSC number and decrease the number of bone marrow stem cells (68). Numerous studies show that GSLs, such as Gb5 and MSGb5, are markers of stem cells; however, no cutting evidence links their expression to a role in pluripotency, and their molecular functions remain unclear. Gb5 and MSGb5, as markers of human ESCs, disappear with differentiation (64, 69). Mass spectrometry analysis that can distinguish glycans shows a switching of the core structures of glycosphingolipids from globo-series and lacto-series to ganglio-series when human ESCs differentiate into embryoid body outgrowth with three germ layers (69). Recent study shows that ganglioside GD2 can be used as a marker to identify CSCs (GD2⁺) from breast cancer cell lines and patient samples, and interference with GD3 synthase can reduce CSC population and CSC-associated properties (70). The present study found GCS and Gb3 overproduced in BCSCs, not normal adult stem cells, such as mammary epithelial stem cells (Fig. 3) and bone marrow stem cells reported by another study (68). It is not clear by which molecular mechanism CSCs regain ability in ceramide glycosylation that exists in ESCs. We now know that ceramide generated in cells exposed to doxorubicin can activate GCS promoter and up-regulate its expression (38).

A major contribution of the present work is that we identified the role of Gb3 in maintaining stem properties of BCSCs. It has been reported that Gb3 (CD77), a precursor of Gb5 is associated with Src family Yes kinase on GSL-enriched microdomains (25, 71). Gb3 can modulate c-Src kinase in GSL-enriched microdomains and up-regulate mdr1 expression through β-catenin signaling (25). Wnt/β-catenin signaling maintains hematopoietic stem cells and neuronal stem cells and the epithelial to mesenchymal transition for BCSCs (72); Wnt inhibitors can reduce the size of tumor spheres and self-renewal in prostate cancer stem cells (73). FGF-2 is critical for the self-renewal and maintenance of normal and cancer stem cells (47, 48), and Oct-4 is a transcription factor that is involved in maintaining the pluripotency of ESCs (74) and BCSCs (59, 75). This study has found that the levels of GCS, Gb3, phosphorylated c-Src, β-catenin, FGF-2, and Oct-4 are substantially higher in BCSCs (Fig. 7). Stepwise inhibition of GCS, c-Src kinases, and β-catenin indicates that these molecules are critical for the maintenance of BCSCs (Fig. 7). Although stem cell factors including β-catenin, FGF-2, Oct-4, and CD44 are involved in regulating stemness of either normal stem cells or CSCs, this study demonstrates that GSLs in BCSCs activate c-Src/β-catenin signaling and up-regulate these factors to maintain BCSCs selectively. In addition to insight regulating stemness of stem cells, this finding opens the possibility of disrupting ceramide glycosylation or GSL synthesis to target CSCs specifically.