UDP-glucose ceramide glucosyltransferase activates AKT, promoted proliferation, and doxorubicin resistance in breast cancer cells

Abstract

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.

Electronic supplementary material

The online version of this article (10.1007/s00018-018-2799-7) contains supplementary material, which is available to authorized users.

Introduction

In the year 2015, breast cancer was declared as the second leading cause of cancer death in women in industrial countries [1]. This fact underlines the importance to investigate the fundamental cellular processes in breast (cancer) cells, which are leading, for example, to promoted proliferation and multidrug resistance development. Multidrug resistance of cancer cells is the main cause of therapy failure. It is accomplished by alteration of myriad cellular signaling cascades resulting, for example, in enhanced expression of multidrug resistance proteins, which transport toxic substances out of cancer cells.

The UDP-glucose ceramide glucosyltransferase (UGCG) was first cloned by Ichikawa et al. [2] and is connected to processes of multidrug resistance in cancer cells. This in the cis-Golgi apparatus residing protein transfers a glucose moiety in β-linkage to the position 1 hydroxyl group of ceramide, which results in glucosylceramide (GlcCer) formation (Fig. 1). GlcCer, also named cerebrosides, serve as precursors for all complex glycosphingolipids (GSLs). Ceramides, which are used for GlcCer synthesis, are produced in the endoplasmic reticulum by six mammalian ceramide synthase (CerS) isoforms, which have a substrate specificity for acyl-CoenzymeAs (acyl-CoAs) of defined chain length [3]. Accordingly, each CerS isoform produces ceramide species of a specific chain length. Ceramides are transported to the Golgi apparatus where they are used for the synthesis of sphingomyelin (constituted of ceramide and phosphocholine or a phosphoethanolamine group) or cerebrosides (Fig. 1). Subsequently, cerebrosides can be used as precursors for synthesis of lactosylceramides (LacCer), which are also named globosides. Globosides can be metabolized to gangliosides.

Fig. 1.

Schematic overview of the potential mechanisms of UGCG-derived GSLs influencing membrane lipid composition resulting in cellular signaling pathway induction. Overexpression of UGCG results in increased GlcCer concentration leading to Gb3 accumulation and augmented integration of GlcCer in plasma membrane structures. This results in altered biophysical membrane properties of glycosphingolipid-enriched microdomains (GEMs), which may alter membrane protein activities and activation of signaling pathways like Akt and ERK1/2. Activation of these kinases increases proliferation and MDR1 expression. GSL glycosphingolipid, GEM glycosphingolipid-enriched microdomain, P-gp P-glycoprotein, bis I bisindolylmaleimide I, PKC protein kinase C, PI3K phosphoinositide 3-kinase (PI3K)

Knockout of the UGCG in mice leads to embryonic lethality during the phase of gastrulation [4]. In addition, a constitutive disruption of this protein in mice epidermis results in loss of skin barrier function and death due to dehydration [5]. Amen et al. showed that GlcCer is essential for proper formation of the lamellar body, regular metabolism, and composition of lipids in the stratum corneum [6]. All these parameters are important for maintaining water permeability function. Deletion of the UGCG in nervous system-specific cells leads to disturbance of brain tissue by the loss of Purkinje cells (reviewed in [7]). Moreover, long-term pharmacological inhibition of the UGCG with eliglustat in patients with Gaucher disease type 1 was well tolerated [8].

The UGCG is overexpressed in several cancer types, for example, in metastatic breast cancer tissue resulting in a poor patient prognosis [9] and colon cancer cells [10]. This overexpression correlates with an enhanced expression of P-glycoprotein 1 (P-gp) (also ATP-binding cassette sub-family B member 1, ABCB1), which is encoded by the multidrug resistance protein 1 (MDR1) gene. The exact molecular mechanisms by which UGCG and MDR1 are connected are unknown, but it could be shown that MDR1 regulates UGCG promoter activity as well as UGCG regulates MDR1 expression (reviewed in [11–13]). Liu et al. showed that globo-series GSL produced by UGCG activity alter MDR1 expression [14]. Overexpression of UGCG in combination with chemotherapeutic agents leads to increased Gb3 and Gb5 concentrations in GSL-enriched microdomains (GEM). This results in cSrc tyrosine kinase activation, decreased β-catenin phosphorylation, and increased nuclear β-catenin. It is assumed that nuclear β-catenin may bind in a complex with the T-cell factor 4 (Tcf4) to the Tcf4/lymphoid enhancer factor (LEF) binding motif at the MDR1 promoter and thus enhancing promoter activity. This leads to enhanced P-gp expression and subsequently to efflux of anti-cancer drugs from cells. P-gp is also postulated to function as a flippase, transporting GlcCer from the outer to the inner leaflet of the Golgi apparatus, where it can be metabolized to more complex sphingolipids like GSLs (reviewed in [15]).

The regulation of the UGCG is rarely investigated. Beside CpG island methylation of the UGCG promoter in ductal breast cancer cells, which seems to be important for drug resistance [16], doxorubicin in combination with the estrogen receptor (ER) subtype α leads to an increased UGCG promoter activity possibly mediated via a Sp1-binding site [17]. The UGCG protein activity is influenced by the dimerisation with c-Fos [18] and the neuroendocrine-specific protein Reticulon-1C [19].

Here, we investigated the cellular mechanisms affected by UGCG overexpression in breast cancer cells. Our data indicate that an increased UGCG expression leads to transformation of the cell metabolism including an altered morphology and transcriptional upregulation of MDR1. In addition, anti-apoptotic gene expression is enhanced and pro-apoptotic gene expression reduced. These cellular changes result in a doxorubicin resistance and promoted proliferation of MCF-7/UGCG cells. Therefore, the UGCG is a key enzyme in breast (cancer) cells signaling leading to severe alterations of the cell metabolism.

Results

Establishing a stably UGCG overexpressing MCF-7 cell line

MCF-7 cells were transfected with an UGCG expression plasmid (MCF-7/UGCG OE) or a control vector (MCF-7/pTarget) (MCF-7/naiv = no transfection). After selection of the cells with G418 over several weeks, UGCG mRNA and protein expression was analyzed. Figure 2a shows a significantly increased UGCG mRNA expression in MCF-7/UGCG OE cells as compared to MCF-7/naiv and MCF-7/pTarget cells. This is verified at protein level by Western blot analysis (Fig. 2b). In addition, the UGCG overexpression in MCF-7/UGCG OE cells was confirmed by immunocytochemistry (Fig. 2c). In all three MCF-7 cell types, UGCG co-localizes with GM130, a marker for the cis-Golgi apparatus. In summary, the overexpression of UGCG protein in MCF-7/UGCG cells has been shown.

Fig. 2.

Stable UGCG overexpression in MCF-7 cells. a Expression analysis of UGCG mRNA in MCF-7 cells by qRT-PCR. The expression is related to the housekeeping gene RPL37A. Data are represented as a mean of n = 7–11 ± SEM (standard error of the mean). Unpaired t test with Welch’s correction. b Western blot analysis of MCF-7 cells. Densitometrically evaluated UGCG protein concentration and a representative blot is displayed. Data are represented as a mean of n = 5 ± SEM. Unpaired t test with Welch’s correction. c Immunocytochemistry of MCF-7 cells. Cells were incubated with an anti-UGCG and anti-GM130 (cis-Golgi apparatus) antibody and subsequently incubated with secondary antibodies. DAPI (4′,6-diamidino-2-phenylindole) was used to stain DNA. Images were recorded by Axio Observer. Z1 microscope (Carl Zeiss AG, Oberkochen, Germany). *p ≤ 0.05, **p ≤ 0.01

Morphological and physiological changes following UGCG overexpression

Overexpression of UGCG in MCF-7 cells leads to an enlarged cytoplasm as compared to control cells (Fig. 3a). In addition, MCF-7/UGCG OE cells exhibit an up to 70% increased nucleus size as quantified by calculating the nucleus-to-cytoplasm (N:C) ratio in the different MCF-7 cells (Fig. 3b). This ratio is defined as the ratio of the nuclear area divided by the cytoplasmic area indicating abnormal nuclear morphology. MCF-7/UGCG OE cells exhibit also a promoted proliferation indicated by a fivefold higher living cell number than the control cell number after 5 days of culturing (Fig. 3c). This effect can partially be reversed by pretreatment with PPMP, an UGCG inhibitor, indicating that the UGCG is important for this proliferation promoting effect. The results are confirmed by UGCG knockdown (UGCG KD) experiments (supplemental 1A). MCF-7/UGCG KD cells are smaller as compared to control and MCF-7/UGCG OE cells (supplemental 1B). Staining of the cytoskeleton and cell membrane by β-actin and β-catenin validates the different cell sizes following UGCG overexpression and UGCG knockdown (supplemental 2A). In addition, MCF-7/UGCG KD cells exhibit a significantly reduced living cell number following 5 days of cultivation (supplemental 1C). MCF-7 cells were treated for 48 h with doxorubicin, which is frequently used for chemotherapy in breast cancer patients. MCF-7/UGCG OE cells are less sensitive to doxorubicin than MCF-7 control cells (Fig. 3d). The viability of MCF-7/naiv cells is reduced to 50% at a doxorubicin concentration of 7.75 µM. MCF-7/pTarget cells are 50% reduced in their viability at a doxorubicin concentration of 7 µM, whereas MCF-7/UGCG OE cells are vital up to 50% at a concentration of 60 µM doxorubicin. PPMP treatment increases the doxorubicin sensitivity of MCF-7/naiv cells to 2.6 µM. The doxorubicin sensitivity of MCF-7/pTarget cells remains unaltered following PPMP treatment. MCF-7/UGCG OE cells are resensitivated to doxorubicin by PPMP treatment showed by 50% cell viability at a doxorubicin concentration of 40 µM (Fig. 3d). These data indicate that the UGCG is involved in the process of drug resistance.

Fig. 3.

Morphological and physiological changes following UGCG overexpression in MCF-7 cells. a Transmitted light image acquisition shows an enlarged cytoplasm of MCF-7/UGCG OE cells. b Nucleus-to-cytoplasm ratio of MCF-7 cells. Data are represented as a mean of n = 6 ± SEM. Unpaired t test with Welch’s correction. c Cell proliferation with and without 2 µM PPMP over 5 days. Data are represented as a mean of n = 5–11 ± SEM. Unpaired t test with Welch’s correction. d Cell viability analysis with and without 2 µM PPMP pretreatment over 5 days. Doxorubicin was added for 48 h. Data are represented as a mean of n = 4 ± SEM. Unpaired t test with Welch’s correction. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

UGCG overexpression influences mRNA expression of sphingolipid metabolizing enzymes and MDR1

Overexpression of UGCG in MCF-7 cells has a severe impact on the mRNA expression of several sphingolipid metabolizing enzymes. Figure 4a shows that in MCF-7/UGCG OE cells, the mRNA expression of CerS2, -5, CERK, and SMS1 is significantly increased as compared to MCF-7/pTarget cells. In contrast, the mRNA expression of CerS4, -6, aSMase, nSMase1, and -2 and aCDase are downregulated in these cells (Fig. 4b). In addition, MCF-7/UGCG OE cells exhibit an increased Gb3 synthase mRNA expression as compared to control cells, whereas the GM3 synthase mRNA expression is unaltered following UGCG overexpression (Fig. 4d). To exclude the possibility that the changes in mRNA expression are a monolayer-dependent effect, 3D MCF-7 spheroid assembly was performed, and subsequently, the qRT-PCR analysis repeated (supplemental 3A). In spheroids from MCF-7/pTarget and MCF-7/UGCG OE cells, nearly all enzymes of the sphingolipid pathway were comparably expressed as under monolayer conditions, with respect to the expression of CerS2, CerS5 and SMS1 mRNA, which are decreased in MCF-7 spheroids compared to 2D cultured MCF-7 cells. The difference regarding CerS2, CerS5, and SMS1 mRNA expression is possibly due to increased cell–cell contacts in the 3D spheroid model or due to nutrition shortage. Supplemental 4 shows that MCF-7/UGCG OE spheroids are smaller in size and more densely packed than control cell spheroids. In addition, MCF-7/naiv and MCF-7/pTarget spheroids exhibit cell-free spaces represented by the DAPI staining, which indicates a nutrition deficit in MCF-7/UGCG OE spheroids or a necrotic core of MCF-7/naiv and MCF-7/pTarget spheroids. Hence, the differences in the mRNA expression levels of various enzymes of the sphingolipid pathway occur due to the overexpression of the UGCG gene in these cells and are not a monolayer-dependent effect. In addition, Fig. 4a shows an UGCG-dependent increase of MDR1 mRNA expression. Treatment of MCF-7/UGCG OE cells with 2 µM PPMP, an UGCG inhibitor, 2 µM bisindolylmaleimide I [protein kinase C (PKC) inhibitor], or 25 µM Ly294002 [phosphoinositide 3-kinase (PI3K) inhibitor] for 48 h abolishes the UGCG-mediated increase of MDR1 mRNA expression in MCF-7/UGCG OE cells (Fig. 4c). In contrast, MDR1 mRNA expression of MCF-7/pTarget cells was not affected by treatment with PPMP, Bisindolylmaleimide I, or Ly294002. This is possibly based on the fact that MCF-7/pTarget cells already exhibit a very low basal MDR1 mRNA expression as compared to MCF-7/UGCG OE cells (Fig. 4a). Consistent with these data, MDR1 mRNA expression is decreased in MCF-7/UGCG KD cells as compared to MCF-7/UGCG OE cells and control cells (supplemental 2B).

Fig. 4.

Analysis of mRNA expression of sphingolipid metabolizing enzymes and MDR1 in MCF-7 cells by qRT-PCR. a Anti-apoptotic/pro-proliferative enzymes: MDR1, CerS2, CerS5, CERK, and SMS1. b Pro-apoptotic/anti-proliferative enzymes: CerS4, CerS6, aSMase, nSMase1, nSMase2, and aCDase. c MDR1 mRNA expression following 48 h without stimulation, 2 µM PPMP, 2 µM bisindolylmaleimide I, or 25 µM Ly294002. d Gb3 and GM3 synthase mRNA expression. The mRNA expression is related to the housekeeping gene RPL37A. Data are represented as a mean of n = 3–10 ± SEM. Unpaired t test with Welch’s correction. *p ≤ 0.05, **p ≤ 0.01

The impact of an increased UGCG expression on sphingolipid levels

Strong expression of the UGCG leads to altered levels of several sphingolipid metabolites. First, we analyzed complex sphingolipids by HPTLC analysis, which revealed that MCF-7/UGCG OE cells show lower concentrations of total ceramide and GlcCer, whereas Gb3, a globo-series glycosphingolipid, is strongly increased in its concentration in comparison to MCF-7/pTarget cells (Fig. 5a, lane 1 and 2, b). This is in line with the qRT-PCR analysis showing an increased Gb3 synthase mRNA expression in MCF-7/UGCG OE cells as compared to control cells (Fig. 4d). Figure 5a, b shows also a slight increase in the concentration of gangliosides and more complex GSLs in MCF-7/UGCG OE cells (line 2). Interestingly, also a shift from LacCer with very long chain length to LacCer with long chain length is detectable in MCF-7/UGCG OE cells. PPMP treatment reduces all glycosylated sphingolipids in their concentration in MCF-7/pTarget as well as in MCF-7/UGCG OE cells as shown in Fig. 5a line 3 and 4 and Fig. 5c. The determination of sphingolipid levels by HPTLC is verified and analyzed in more detail by LC–MS/MS (Fig. 5d–f). Figure 5d supports the HPTLC analysis by showing decreased total Cer and GlcCer levels in MCF-7/UGCG OE cells as compared to MCF-7/pTarget cells. This is also shown in Fig. 5e, f. Interestingly, the LC–MS/MS analysis shows an increased LacCer level in MCF-7/UGCG OE cells, which is not detectable by HPTLC. The HPTLC analysis indicates a shift of long-to-very long chain LacCer, which could not be proven by LC–MS/MS due to the lack of standards for the determination of very long chain LacCer species. Figure 5e shows a reduction of C_14:0-, C_18:1-, C_18:0-, and mainly C_24:0-ceramide level in UGCG overexpressing cells. This effect is partially reversed by PPMP pretreatment. PPMP treatment clearly reduced C_16:0, C_24:0-, and C_24:1-GlcCer in both cell lines (Fig. 5f), which verifies the HPTLC analysis. Supplemental 5A shows a decrease of the sphingosine-1-phosphate (Sph-1p) concentration following UGCG overexpression as compared to control cells, which may be the outcome of an accelerated ceramide metabolism to complex sphingolipids. Stimulation with PPMP leads in MCF-7/pTarget and MCF-7/UGCG OE cells to a light reduction of Sph-1p, which is not significant. To investigate whether or not sphingolipid levels following UGCG overexpression exhibit the same pattern in MCF-7/UGCG OE monolayer cultured cells as in MCF-7/UGCG OE spheroids, the LC–MS/MS analysis was repeated for MCF-7/UGCG OE spheroids. Our data show the same alterations of the sphingolipid levels in MCF-7/UGCG OE spheroids as in monolayer cultured cells (supplemental 3B–D). However, LacCer species are slightly reduced in MCF-7/UGCG OE spheroids as compared to monolayer conditions. In summary, our data indicate that an increased UGCG expression leads to an enhanced formation of Gb3 and gangliosides and more complex GSLs in MCF-7 cells, which is accompanied by a reduction of total Cer, GlcCer, and Sph-1p concentration (Fig. 1).

Fig. 5.

Determination of sphingolipid concentrations in MCF-7 cells by LC–MS/MS and HPTLC analysis. a Representative HPTLC plate showing the separation of ceramides, DAG, non-complex GSLs like GlcCer, LacCer, and complex GLSs like Gb3 and gangliosides and more complex GSLs. b Densitometrical analysis of the HPTLC plate lines 1 and 2 (control). c Densitometrical analysis of the HPTLC plate lines 3 and 4 (treatment with 2 µM PPMP for 48 h). All given analytes were related to the respective ceramide spot. d Total Cer, Glc-, and LacCer levels determined by LC–MS/MS. e Concentrations of C_14:0-, C_16:0-, C_18:1-, C_18:0-, C_20:0-, C_24:0-, and C_24:1-Cer determined by LC–MS/MS f. Concentrations of C_16:0-, C_24:0-, and C_24:1-GlcCer, and C_16:0-, C_24:0-, and C_24:1-LacCer determined by LC–MS/MS. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001

UGCG overexpression alters the composition of glycosphingolipid-enriched microdomains (GEMs)

Next, we investigated whether or not the alterations in the sphingolipid composition have an impact on the formation of lipid domain structures in cellular membranes. Therefore, we isolated GEMs by sucrose gradient centrifugation. The lipid content of the separated sucrose fractions was subsequently analyzed by LC–MS/MS. Fraction 2 of MCF-7/UGCG OE cell membranes exhibits a significant increase of GlcCer content in comparison to MCF-7/pTarget cell membrane fraction 2 (Fig. 6a). In contrast, if we sum up the GlcCer concentration of all fractions, the total GlcCer content is decreased slightly in MCF-7/UGCG OE cells as compared to MCF-7/pTarget cells (Fig. 6b), which is in line with the LC–MS/MS and HPTLC data showing a loss of GlcCer (Fig. 5). Supplemental 5B shows that also the total sphinganine and total ceramide levels are increased in fraction 2, whereas total LacCer is slightly decreased. Particularly, the concentrations of C_14:0-, C_16:0-Cer, and C_16:0-, and C_18:1-GlcCer are increased in fraction 2 of MCF-7/UGCG OE cells as compared to MCF-7/pTarget cells (data not shown). Further analysis revealed out that fractions 2 and 3, which are enriched in GlcCer, contain the highest cholesterol and caveolin-1 content (Fig. 6c). Cholesterol and caveolin-1 are markers for GEMs. These fractions could also be defined as the lipid fractions, since the protein content is low, but the sodium potassium ATPase (SPATPase), a plasma membrane protein, is clearly detectable (supplemental 6). Figure 6d shows the sphingomyelin (SM) concentration in whole-cell lipid extracts from MCF-7/pTarget and MCF-7/UGCG OE cells. UGCG overexpression has no effect on SM, respectively; further proceeding of ceramides to sphingomyelin can be excluded. Our data indicate that MCF-7/UGCG OE cells show an alteration of the GEM composition in their membranes as compared to control cells. GEMs are known to be signaling platforms for several membrane-associated proteins [20, 21]. Accordingly, UGCG overexpression might affect membrane-associated proteins and cellular signaling pathways, which is debated in the “Discussion”.

Fig. 6.

Determination of sphingolipids in glycosphingolipid-enriched microdomains (GEMs) of MCF-7/cells. a GEMs were isolated and total GlcCer levels of fractions 1–10 were determined by LC–MS/MS. Data are represented as a mean of n = 3 ± SEM. Unpaired t test with Welch’s correction. b GEMs were isolated and total GlcCer levels of fractions 1–10 were determined by LC–MS/MS. Results of fraction 1–10 are summarized. Data are represented as a mean of n = 3 ± SEM. c Cholesterol-level determination by ELISA of fraction 1–5. Data are represented as a mean of n = 3 ± SEM. Caveolin-1 protein content determined by Western blot analysis. Data are represented as a mean of n = 3 ± SEM. One representative blot of three is displayed. d Sphingomyelin concentration determination of whole-cell lipid extract by LC–MS/MS. SM = Sphingomyelin. Data are represented as a mean of n = 3 ± SEM. *p ≤ 0.05, **p ≤ 0.01

Effect of UGCG overexpression on signaling cascades in MCF-7 cells

To reassess our hypothesis that MCF-7/UGCG OE cells exhibit an altered composition of their signaling platforms in the cellular membranes, we analyzed various signaling pathways using the Pathscan^® Intracellular Signaling Array Kit. The antibody-based analysis of the phosphorylation status of various key signaling proteins shows an increased phosphorylation of Akt308, glycogen synthase kinase 3β (GSK-3β), Bad, proline-rich Akt substrate of 40 kDa (PRAS40), ribosomal protein S6 (rpS6), and extracellular signal-regulated kinase 1/2 (ERK1/2) in MCF-7/UGCG OE cells compared to MCF-7/pTarget cells (Fig. 7). Activation of these signaling pathways is abolished by PPMP pretreatment (supplemental 7). The antibody-based analysis was repeated for 3D MCF-7/UGCG OE spheroids and shows also a positive phosphorylation status of Akt308, PRAS40, rpS6, and ERK1/2 in the 3D cell culture (supplemental 8). In addition, data were confirmed with MCF-7/UGCG KD cells (supplemental 9), which verify an UGCG-dependent phosphorylation of Akt308, GSK-3β, Bad, PRAS40, rpS6, and ERK1/2. In summary, our data indicate that in MCF-7/UGCG OE cells, several signaling pathways are induced, which might contribute to the observed cellular changes such as increased cell proliferation and doxorubicin drug resistance (Fig. 1).

Fig. 7.

Analysis of phosphorylation status of key signaling molecules in MCF-7 cells by an antibody-based array. Array was performed according to the manufacturer’s protocol. a Densitometrical analysis of the array. Data are represented as a mean of n = 5–7 ± SEM. b Representative arrays were displayed. RFU relative fluorescence unit. *p ≤ 0.05

Discussion

We were able to show that an increased UGCG mRNA expression in MCF-7 cells leads to cellular changes such as an altered enlarged cytoplasm, which promoted proliferation and doxorubicin resistance. These effects are abolished by pretreatment of the cells with PPMP, an UGCG inhibitor, and UGCG knockdown, indicating that these effects are UGCG-mediated.

Our LC–MS/MS analysis shows a loss of Cer and GlcCer and a slight increase of LacCer. The accumulation of Gb3 and gangliosides in UGCG overexpressing MCF-7 cells indicates that GlcCer are metabolized immediately to LacCer and subsequently to complex GSLs, especially Gb3. This is accomplished by an increased Gb3 synthase mRNA expression in MCF-7/UGCG OE cells. The GM3 synthase mRNA expression remains unaltered indicating a transcriptional-independent regulation. Possibly, the GM3 synthase exhibits an increased enzyme activity due to altered membrane composition following UGCG overexpression resulting in GM3 accumulation. The reduction of C_24:0-Cer and C_24:0-GlcCer in MCF-7/UGCG OE cells indicates that preferably ceramides with a chain length of C_24:0 are further metabolized to more complex GSL in UGCG overexpressing cells. This result is in line with studies from Yamaji et al., who investigated the acyl chain length of sphingomyelin and GSLs in HeLa cells, which expressed variable amounts of CerS2. By performance of metabolic labeling experiments, they could show that preferentially very long chain ceramides such as C_24:0-Cer are transported to the UGCG via a thitherto unknown mechanism [22]. This study showed also that C_16:0-Cer is predominantly used for sphingomyelin synthesis, which seems also to be true in our study showing only a slight decrease of C_14:0-, C_16:0-, C_18:1-, and C_18:0-Cer in MCF-7/UGCG OE cells. However, although the GlcCer concentrations decrease following UGCG overexpression our data indicate that the remaining GlcCer are integrated in the cell membrane leading to an enrichment of membrane structures with high GlcCer content in MCF-7/UGCG OE cells as compared to control cells. However, Sph-1p levels are decreased in MCF-7/UGCG OE cells as compared to control cells, which are contradictory to the concept of sphingolipid rheostat, which claims the pro-proliferative character of Sph-1p and an anti-proliferative character of Cer (reviewed in [23]). However, the role of Sph-1p in cellular processes is still under investigation and controversial (reviewed in [24]). The lowered Sph-1p level following UGCG overexpression may be the consequence of an accelerated sphingolipid metabolism resulting in accumulation of complex GSL. However, further analysis of the fractionated cellular lysates by sucrose gradient centrifugation revealed that the fractions 2 and 3, which contain increased GlcCer concentrations, also contain the highest cholesterol and caveolin-1 level. Caveolins are one of the main constitutes of GEM, respectively rafts and are used as raft markers [25, 26]. The data identify fractions 2 and 3 as glycosphingolipid-enriched microdomains (GEMs) localized in the plasma membrane of MCF-7/pTarget and MCF-7/UGCG OE cells. Whether or not the accumulation of Gb3 or changes in another lipid is responsible for altered membrane protein activity and, therefore, leading, for example, to promoted proliferation needs to be investigated. It is known that GEMs are signaling platforms for several membrane-associated proteins [20, 21]. This leads to the assumption that UGCG overexpression might affect membrane proteins and subsequently cell signaling pathways. An example for this might be the epidermal growth factor receptor (EGFR), which is connected to promoted MCF-7 cell proliferation. Rogers et al. showed that EGFR signaling in cancer cells is reliant on the localization of EGFR in intact lipid rafts [27]. Disruption of the receptor localization leads to decreased Ras activation and subsequent downregulation of ERK signaling resulting in proliferation inhibition. Investigating the activation of key signaling proteins in MCF-7/UGCG OE cells revealed a positive phosphorylation status of the key signaling proteins Akt308, GSK-3β, Bad, PRAS40, rpS6, and ERK1/2, indicating that overexpression of the UGCG activates anti-apoptotic and pro-proliferative signaling pathways. We assume that the altered composition of the GEMs leads to the phosphorylation of Akt and ERK1/2 in MCF-7/UGCG OE cells. The serine/threonine kinase Akt is phosphorylated at threonine (T) 308, which is sufficient for its activation. How Akt is phosphorylated exactly and, therefore, activated needs to be investigated in future studies. Akt308 is known to phosphorylate GSK-3β and Bad leading to an inhibition of both proteins resulting in promoted cell survival and growth (reviewed in [28]) (Fig. 1). Akt308 phosphorylation also leads to PRAS40 phosphorylation, which negatively regulates mTOR activity by binding directly to the mTORC1 complex. Phosphorylated PRAS40 is inactive and dissociates from the mTORC1 complex leading to an activation of the mTOR signaling pathway. Activation of the mTOR signaling pathway is also indicated by a positive phosphorylation status of rpS6, which is a downstream target of mTOR, leading, for example, to cell cycle progression and subsequently to accelerated cell growth (reviewed in [28]). Beside Akt, the ERK1/2 signaling pathway is induced, which may also contribute to promoted proliferation (reviewed in [29]) (Fig. 1). Treatment of MCF-7/UGCG OE cells with PPMP, an UGCG inhibitor, and UGCG knockdown abolishes the phosphorylation of Akt, GSK-3β, Bad, PRAS40, rpS6, and ERK1/2 significantly indicating an UGCG-dependent activation of these signaling pathways. Since the mTORC1 complex regulates translation initiation and ribosome biogenesis, perfect conditions for an increased gene expression of anabolic genes are given when the mTOR pathway is activated like it is assumed for MCF-7/UGCG OE cells. Accordingly, genes, which encode for cell metabolism regulator proteins, are altered in its expression in the way that cell proliferation is intensified (Table 1) (CerS2 [30, 31], CerS5 [32], CERK [33], SMS1 [34–36], CerS4 and -6 [37], CerS6 (reviewed in [3], aSMase (reviewed in [38], nSMase1 [39], nSMase2 [40–42], and aCDase [43]).

Table 1.

Summary of mRNA expression analysis data of MCF-7 cells

Protein	MCF-7/UGCG cells compared to control cells	Function	References
MDR1	↑	Multidrug resistance ↑	[9]
CerS2	↑	Colony formation ↑ Anti-apoptotic	[30] [31]
CerS5	↑	Autophagy ↑	[32]
CERK	↑	Tumor cell survival ↑ Mammary tumor recurrence ↑	[33] [33]
SMS1	↑	Proliferation ↑ Anti-apoptotic Regulation of raft structures (signaling platforms)	[34] [35] [36]
CerS4	↓	Colony formation ↓ Proliferation ↓	[37] [37]
CerS6	↓	Colony formation ↓ Proliferation ↓ Pro-apoptotic	[37] [37] [3]
aSMase	↓	Pro-apoptotic	[38]
nSMase1	↓	Pro-apoptotic	[39]
nSMase2	↓	Pro-apoptotic ↓ Cell viability ↓ Growth rate ↓	[40] [41] [42]
aCDase	↓	Tumor recurrence ↓	[43]

The protein name and the alterations regarding mRNA expression of MCF-7/UGCG OE cells compared to MCF-7/pTarget cells are given. In addition, the function and the respective reference are denoted

Expression analysis of CerS2, -5, and CERK in spheroids shows a less expression in MCF-7/UGCG OE cells compared to MCF-7/pTarget cells. We could show that MCF-7/UGCG OE spheroids are packed more densely; accordingly, the alterations in mRNA expression may be contributed to a deficit of energy supply in the core of MCF-7/UGCG OE spheroids. In vivo these conditions would lead to angiogenesis. Cancer cells might stimulate angiogenesis by secretion of Sph-1p [44], whereas the Sph-1p level in the spheroids in our study was not detectable (data not shown). The cell-free area in MCF-7/naiv and MCF-7/pTarget spheroids could also be an indicator for the development of a necrotic core of the spheroid, which seems not to be induced in MCF-7/UGCG OE spheroids. However, the cell-free core of MCF-7/naiv and MCF-7/pTarget spheroids might be a necrotic core, which is lacking in MCF-7/UGCG OE spheroids. Since 3D MCF-7/UGCG OE spheroids show similar data, we assume that the UGCG effect seems not to be monolayer-dependent. However, MCF-7/UGCG OE cells exhibit high proliferation rate despite the limited nutrient availability leading to the assumption that the UGCG has an influence on energy metabolism or at least on the efficacy of energy uptake out of the media. The data indicate that MCF-7/UGCG OE cells alter their metabolisms in the way that anabolic processes are maintained and catabolic mechanisms are restricted resulting in the described cellular impacts. MCF-7/UGCG OE cells may achieve these effects by induction of autophagic processes. Shen et al. showed an UGCG inhibition-dependent autophagic flux in neurons [45]. Furthermore, CerS5 mRNA is increased in MCF-7/UGCG OE cells, which has also been associated with autophagy. Gosejacob et al. showed that adipose tissue of CerS5 knockdown mice exhibits less autophagy following high fat diet [46]. Because of the doxorubicin resistance of MCF-7/UGCG OE cells, we investigated the mRNA expression of the multidrug resistance protein 1 (MDR1) in MCF-7 cells. The basal mRNA expression of MDR1 is strongly increased in MCF-7/UGCG OE cells indicating the mediation of the doxorubicin resistance by MDR1. It is already shown that an UGCG overexpression is accompanied by an increased MDR1 gene expression in several cancer cell types [9] and that MDR1, which encodes the protein P-glycoprotein 1 (P-gp) (also ATP-binding cassette sub-family B member 1, ABCB1), which leads to multidrug resistance, for example, against vinblastine. By blocking, the UGCG resensitization of multidrug-resistant breast cancer cells to anti-cancer drugs via downregulation of MDR1 has been achieved [10]. The induction of drug resistance to cytotoxic agents is accomplished by highly complex mechanisms and is the main cause of anti-cancer therapy failure. In MCF-7 cells, inhibition of the sphingosine kinase-2 (SphK2), which produces Sph-1p, resensitizes the cells to standard chemotherapy [47], but, as mentioned above, MCF-7/UGCG OE cells exhibit a decreased Sph-1p level as compared to control. The P-gp protein expressed at the plasma membrane transports cytotoxic substances out of the cell (reviewed in [48]) (Fig. 1). However, this efflux pump seems also to play a role in the Golgi apparatus. The UGCG protein executes its task of ceramide to GlcCer conversion on the cytosolic surface of the Golgi apparatus. For LacCer synthesis, GlcCer must be flipped to the luminal leaflet of the Golgi apparatus. Morad and Cabot postulate that P-gp flips GlcCer from the Golgi apparatus cytosol to the Golgi lumen, thereby contributing to multidrug resistance (reviewed in [15]). This contribution is supported by the fact that beside ceramides, GlcCer induces toxic effects in the cell [49], and by flipping GlcCer, it is used for non-toxic complex GSL synthesis. Interestingly, the presence of cholesterol, which influences membrane packing and fluidity and which is increased in fraction 2 and 3 in MCF-7 cells, influences P-gp activity (reviewed in [50]). Since the cholesterol levels in MCF-7/pTarget and MCF-7/UGCG OE cells are similar, it should be investigated whether or not the combination of cholesterol and GlcCer is more important for P-gp activity. Interestingly, treatment with a PKC (bisindoylmaleimide I) or PI3K (Ly294002) inhibitor or PPMP could abolish the effect, indicating that downstream of UGCG expression molecular mechanisms leads to an induction of MDR1 mRNA expression. A direct binding of the transcription factor nuclear factor (NF) ΚB to the MDR1 promoter results in decreased MDR1 gene and P-gp protein expression [51]. Liu et al. showed that by UGCG activity generated GSLs, MDR1 expression is mediated by signaling of the tyrosine kinase cSrc and β-catenin [14]. In this case, UGCG overexpression in combination with cytotoxic agents leads to increased globo-series GSLs in GEMs; and as a result, cSrc is activated and nuclear β-catenin increased. A co-binding of β-catenin and the T-cell factor 4 (Tcf4) to the Tcf4/lymphoid enhancer factor (LEF)-binding motif at the MDR1 promoter is assumed. The consequence is an enhanced MDR1 promoter activity [14]. However, the exact molecular mechanisms, which follow an UGCG overexpression and lead to MDR1 gene expression induction, should be investigated in the future.

In conclusion, UGCG overexpression in human breast cancer cells alters the lipid composition of the plasma membrane by increasing the integration of GlcCer resulting in Akt and ERK1/2 activation. This leads to induction of MDR1 and enhanced anti-apoptotic gene transcription as well as reduced transcription of pro-apoptotic genes. Thus, an enhancement of UGCG in tumor cells influences several cellular signaling pathways leading to an altered phenotype, promoted proliferation, and chemotherapy resistance.

Materials and methods

Cell culture

The human breast adenocarcinoma cell line MCF-7 was purchased from the Health Protection Agency (European Collection of Cell Cultures, ECACC, Salisbury, UK). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing high glucose, no phenol-red and no HEPES, 1% GlutaMAX, 1% sodium pyruvate, and 5% charcoaled fetal bovine serum (FBS) (Sigma-Aldrich, Deisenhofen, Germany). Cells were incubated at 37 °C in an atmosphere containing 5% CO₂. For selection of stably transfected cells, G418 (Thermo Fisher Scientific, Waltham, MA, USA) was added.

Cell treatment

MCF-7 cells were seeded at a proper density for the appropriate dish. Because of the high proliferation rate, MCF-7/UGCG OE cells were seeded in a 50% lower density compared to MCF-7/pTarget cells. Cells were stimulated with 2 μM dl-threo-1-phenyl-2-palmitoyl-amino-3-morpholino1-propanol (PPMP) (Enzo Life Sciences, Farmingdale, NY, USA) over 6 days and media were renewed every 48 h. For MDR1 gene expression analysis, MCF-7 cells were treated with 0 and 2 µM bisindolylmaleimide I [Protein kinase C (PKC) inhibitor] (Cayman, Ann Arbor, MI, US) and 0 and 25 µM Ly294002 [Phosphoinositide 3-kinase (PI3K) inhibitor] (Cell Signaling, Cambridge, UK) for 48 h.

Stable transfection with a UDP-glucose ceramide glucosyltransferase (UGCG) expression plasmid

UDP-glucose ceramide glucosyltransferase (UGCG) expression plasmid (pCMV6-ENTRY vector) was purchased from OriGene Technologies Inc. (Rockville, USA). The pTarget empty control vector was purchased from Promega GmbH (Mannheim, Germany). Stable transfection was performed with Lipofectamine^® 2000 Reagent (Invitrogen by Life Technologies) according to the manufacturer’s protocol. MCF-7 cells were transfected with 2 μg of the distinct plasmid and selected over 5 weeks with different G418 concentrations. 200 μg/mL G418 was used for selection of stable transfected cells.

Determination of mRNA expression of MDR1 and sphingolipid metabolizing proteins by quantitative real-time PCR (qRT-PCR)

Total RNA was isolated with RNeasy Mini Kit (Qiagen N.V., Venlo, The Netherlands) according to the manufacturer’s protocol. RNA concentrations were determined photometrical by using the Infinite^® 200 NanoQuant monochromator (Tecan Group, Männerdorf, Switzerland). The cDNA was synthesized from 300 ng total RNA using the VERSO™ cDNA Kit (Thermo Fisher Inc., Waltham, MA, USA). Gene-specific PCR products were assayed using 5× QPCR Mix EvaGreen^® (ROX) (Bio&Sell, Feucht bei Nürnberg, Germany) on a 7500fast quantitative PCR system (TaqMan^®, Life Technologies, Darmstadt, Germany). Relative gene expression was determined using the comparative ∆CT (cycle threshold) method, normalizing relative values to the expression level of 60S ribosomal protein L37a (RPL37A) as a housekeeping gene. Primers for CerS2, -4, -5, and -6 were synthesized by Biomers (Ulm, Germany) and primer for UGCG, acid sphingomyelinase (aSMase), neutral sphingomyelinase 2 (nSMase2), acid ceramidase (aCDase), MDR1, GM3 synthase, and RPL37A were synthesized by Eurofins Genomics (Ebersberg, Germany) (Table 2). The forward and reverse primer sets for neutral sphingomyelinase 1 (nSMase1), ceramide kinase (CERK), and Gb3 synthase were purchased From RealTimePrimers (Elkins Park, Philadelphia, USA). From GeneCopoeia (Rockville, MD, USA), the primer set for sphingomyelin synthase 1 (SMS1) mRNA detection was purchased.

Table 2.

Oligonucleotides for qRT-PCR

Protein	5′-forward-3′	5′-reverse-3′	Amplicon (bp)
CerS2	cca ggt aga gcg ttg gtt	cca ggg ttt atc cac aat gac	142
CerS4	ctg gtg gta cct ctt gga gc	cgt cgc aca ctt gct gat ac	248
CerS5	caa gta tca gcg gct ctg t	att atc tcc caa ctc tca aag a	123
CerS6	aag caa ctg gac tgg gat gtt	aat ctg act ccg tag gta aat aca	146
UGCG	tgc tca gta cat tgc cga aga	tgg aca ttg caa acc tcc aa	74
aSMase	cac cca gga tga gaa tgg aaa	gtc cgt cct cac cca cga t	59
nSMase2	caa caa gtg taa cga cga tgc c	cga ttc ttt ggt cct gag gtg t	89
aCDase	tgt gga tag ggt tcc tca cta ga	ttg tgt ata cgg tca gct tgt tg	375
MDR1	ctc aga cag gat gtg agt tgg t	aca gca agc ctg gaa cct at	116
Gb3 synthase	Tac ctg gac acg gac ttc at	Gga tgg aac acc act tct tg	226
GM3 synthase	Gaa ctc ttg cca gag cac ga	Ccc agt tct aat ccg tgc ag	104
RPL37A	att gaa atc agc cag cac gc	agg aac cac agt gcc aga tcc	94

The protein name, sequence, and amplicon size in bp are given

Protein concentration determination by Western blot analysis

For protein concentration analysis by Western blot, total protein was isolated. Cells were resuspended in the following buffer: 10 mM Tris (pH  8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, and 1% 100X Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, Darmstadt, Germany). The lysate was sonicated and centrifuged (14,000×g, 10 min, 4 °C). For determination of total protein concentration, the Bradford method was performed [52]. 60 μg total protein extract was electrophoretically separated by 12% sodium dodecyl sulfate (SDS)-PAGE and electroblotted onto a nitrocellulose membrane (GE Healthcare Life Sciences, Amersham, UK). After 1.5 h incubation of the membrane in Odyssey^® Blocking Buffer (LI-COR Biotechnology, Lincoln, NE, USA) and 1× PBS (1:1), the membrane was incubated with the respective primary antibodies: anti-UGCG antibody (Abcam, Cambridge, UK) and Hsp90 (BD Bioscience, Heidelberg, Germany). Anti-UGCG antibody was incubated overnight, while the Hsp90 antibody was incubated for 60 min. The fractions, which are described in “Isolation of glycosphingolipid-enriched microdomains (GEMs)”, were incubated after SDS-PAGE with anti-caveolin-1 (Abcam, Cambridge, UK) primary antibody to verify GEMs. For supplemental 9, membranes were incubated with anti-SPATPase, anti-Hsp90, anti-PDI, and anti GAPDH primary antibodies (Abcam, Cambridge, UK). The membranes were analyzed on the Odyssey^® infrared scanner from LI-COR (Bad Homburg, Germany).

Immunocytochemistry

MCF-7/naiv, MCF-7/pTarget, and MCF-7/UGCG OE cells were fixed in 4% paraformaldehyde, blocked with 5% Odyssey^® Blocking Buffer (LI-COR Biotechnology, Lincoln, NE, USA), and incubated over night with anti-UGCG and anti-GM130, anti-β-actin, or anti-β-catenin (Abcam, Cambridge, UK) primary antibody. Subsequently, cells were incubated with fragment cy3- (Sigma-Aldrich, St. Louis, Missouri, USA) and Alexa Fluor^® 488- (Life Technologies, Carlsbad, CA, USA) conjugated secondary antibodies and examined with an Axio Observer.Z1 microscope (Carl Zeiss AG, Oberkochen, Germany).

Sphingolipid concentration determination by high-performance thin-layer chromatography (HPTLC)

All chemical reagents were of analytical grade or higher. Lipid standards were from Avanti Polar Lipids (Alabaster, USA) or Matreya LLC (Pleasant Gap, USA). Organic solvents were from Rathburn Chemicals Ltd. (Walkerburn Scotland). The high-performance thin-layer chromatography (HPTLC) silica plates were from Whatman, UK. 9 × 10⁶ MCF-7 cells were scraped in 1 x PBS, centrifuged, and frozen in liquid nitrogen. Cells were dried under the hood for 3 days and stored in a freezer (− 80 °C) until analyzed. Each dried sample was solubilized in ice cold MQ-water (1 mL) by bath sonication (ice-water bath) for 5 min. The solubilized samples were transferred to clean glass test tubes (with screw caps). Total lipids were extracted by a modified Blight and Dyer protocol: 1 mL of MQ-water was added to each sample (2 mL of total water volume), 3 mL of chloroform:isopropanol (2:1, v:v) was added to each sample, and samples were vortexed thoroughly for 10 s, and rotated end-over-end for 20 min at RT. The samples were centrifuged at 4000 rpm for 20 min at RT to separate the phases. The organic phase (bottom) was carefully extracted with a glass Pasteur pipette and placed in a clean glass tube, without disturbing the protein precipitate at the interphase. The chloroform:isopropanol (2:1, v:v) extractions were repeated once, as described above, and combined with the previous extracts. 3 mL of hexane was then added to each sample, and samples were vortexed, rotated, and centrifuged as described above. The hexane phases (top) were extracted and combined with the previous chloroform:isopropanol extracts. The organic solvent containing the extracted lipids was dried under a stream of nitrogen. The dry samples were stored at − 20 °C. Each of the dried lipid sample was re-solubilized in an appropriate volume of chloroform:isopropanol (2:1, v:v). The lipid samples were normalized according to their corresponding protein concentrations and applied on an HPTLC plate using an autosampler. The HPTLC plate was developed using the solvent system chloroform:methanol:acetone:acetic acid:water (10:2:4:2:1). The sugar residues of the glycolipids were visualized by spraying the plate with an orcinol solution (0.3% orcinol in 20% sulphuric acid) and by heating (< 5 min, 120 °C). Lipids were identified with the help of commercial standards that were run in parallel with the samples on the HPTLC plate.

The precipitated protein was isolated as follows: 1 mL of chloroform and 1.5 mL of methanol were added to the remaining aqueous phases of the samples. Samples were briefly vortexed and centrifuged at 4000 rpm for 20 min. The aqueous upper phase was carefully removed with a glass Pasteur pipette and discarded, leaving the interphase (with the precipitated proteins) untouched. 2 mL of methanol was added to each of the samples. Samples were again briefly vortexed and centrifuged at 4000 rpm for 20 min, to pellet the precipitated proteins. The chloroform:methanol solution was carefully removed and discarded with a glass Pasteur pipette, without disturbing the pelleted proteins. The pellets were carefully dried to completion, under a stream of nitrogen. Each protein sample was re-solubilized in 500 µL of an 8 M urea solution (8 M urea in PBS, pH 6.8, 0.5% SDS) by vigorous vortexing at RT. The protein samples were stored at − 20 °C. The concentrations of the solubilized proteins were determined by the method of Lowry.

Sphingolipid concentration determination by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Cell pellets were resuspended in 150 µL water, while 50 or 200 µL cell culture supernatant was diluted with 100 µL water. Samples were mixed with 150 µL extraction buffer (citric acid 30 mM, disodium hydrogen phosphate 40 mM), and 20 µL of the internal standard solution containing sphingosine-d7, sphinganine-d7 (200 ng/mL each), sphingosine-1-phosphate-d7, C17:0 Cer, C16:0 Cer-d31, C18:0 Cer-d3, C17:0 LacCer, C18:0 DHC-d3, C16:0 LacCer-d3, C18:0 GluCer-d5 (all avanti polar lipids, Alabaster, USA), and C24:0 Cer-d4 (Chiroblock GmbH, Bitterfeld-Wolfen) (400 ng/mL methanol each). The mixture was extracted once with 1000 µL methanol/chloroform/hydrochloric acid (15:83:2, v/v/v). The lower organic phase was evaporated at 45 °C under a gentle stream of nitrogen and reconstituted in 100 µL of tetrahydrofuran/water (9:1, v/v) with 0.2 formic acid and 10 mM ammonium formate. Afterwards, amounts of sphingolipids were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). An Agilent 1100 series binary pump (Agilent technologies, Waldbronn, Germany) equipped with a Luna C8 column (150 mm × 2 mm ID, 3 μm particle size, 100 Å pore size; Phenomenex, Aschaffenburg, Germany) was used for chromatographic separation. The column temperature was 35 °C. The HPLC mobile phases consisted of water with 0.2% formic acid and 2 mM ammonium formate (mobile phase A) and acetonitrile/isopropanol/acetone (50:30:20, v/v/v) with 0.2% formic acid (mobile phase B). For separation, a gradient program was used at a flow rate of 0.3 mL/min. The initial buffer composition 55% (A)/45% (B) was held for 0.7 min and then within 4.0 min linearly changed to 0% (A)/100% (B) and held for 13.3 min. Subsequently, the composition was linearly changed within 1.0 min to 75% (A)/25% (B) and then held for another 2.0 min. The total running time was 21 min and the injection volume was 15 μL. To improve ionization, acetonitrile with 0.1% formic acid was infused post-column using an isocratic pump at a flow rate of 0.15 mL/min. After every sample, sample solvent was injected for washing the column with a 12 min run. The MS/MS analyses were performed using a triple quadrupole mass spectrometer API4000 (Sciex, Darmstadt, Germany) equipped with a Turbo V Ion Source operating in positive electrospray ionization mode. The MS parameters were set as follows: Ionspray voltage 5500 V, source temperature 500 °C, curtain gas 30 psi, collision gas 12 psi, nebulizer gas 40 psi, and heating gas 60 psi. The analysis was done in Multiple Reaction Monitoring (MRM) mode with a dwell time of 20 ms for all analytes. Data acquisition was done using Analyst Software V 1.6 and quantification was performed with MultiQuant Software V 3.0 (both Sciex, Darmstadt, Germany), employing the internal standard method (isotope dilution mass spectrometry). Variations in accuracy of the calibration standards were less than 15% over the whole range of calibration, except for the lower limit of quantification, where a variation in accuracy of 20% was accepted.

Determination of sphingomyelin concentrations by liquid chromatography–tandem mass spectrometry LC–MS/MS

For sphingomyelin concentration determination, MCF-7 cell pellets were processed according to Peng et al. and determination of the analytes was established, respectively [53].

Proliferation assay

MCF-7/naiv, MCF-7/pTarget, and MCF-7/UGCG OE cells were seeded with 1 x 10⁶ cells/5 mm-dish and harvested on day 5 of stimulation, and the living cell number was determined with trypan blue and a Neubauer counting chamber.

Cell viability assay

MCF-7/naiv, MCF-7/pTarget, and MCF-/UGCG OE cells were seeded in 96-well plates and stimulated as mentioned in “Cell treatment”. Cells were treated with 0, 0.5, 4, 8, and 80 µM doxorubicin for 48 h. Subsequently, media were renewed with G418-free media. Water soluble tetrazolium (WST)-1 reagent (Roche Diagnostics, Rotkreuz, Switzerland) was added 1:10 and cells were incubated for 90 min at 37 °C. Absorbance was measured by Infinite^® 200 PRO reader (Tecan Group, Männerdorf, Switzerland) at a wavelength of 450 nm and 620 nm as reference.

Nuclear-to-cytoplasm (N:C) ratio measurement

The nuclear-to-cytoplasm (N:C) ratio is defined as the ratio of the nucleus area (A_N) to the area of the cytoplasm (A_C) (A = π × r²). The radius (r) was calculated by measuring the horizontal and vertical length of the nucleus and the cytoplasm and the average of both values was divided by the factor of 2. By the AxioVision software (Carl Zeiss AG, Oberkochen, Germany), the cytoplasm and the nucleus area were determined and, subsequently, the ratio calculated.

Isolation of glycosphingolipid-enriched microdomains (GEMs)

12 × 10⁶ MCF-7 cells were washed with DPBS twice and scraped. The cell pellet was processed in 566 μL MES buffer [0.15 M NaCl, 0.025 M MES, 1% Triton-X 100, 1× Roche Complete (7×)]. Samples were sonified five times for 20 s (level 3, output 3%), mixed with 1.134 μL 67.5% saccharose solution, and transferred to a saccharose density centrifugation tube. Samples were overlayed with 1.7 mL 37% saccharose solution and subsequently overlayed with 850 μL 5% saccharose solution. Centrifugation at 32.000 rpm at 4 °C for 20 h followed. Fractions each with 450 μL were removed. Fraction 10 was resuspended in 450 μL MES buffer and sonified (three times for 10 s at level 3 and output 3%) because of its viscous nature. 50 μL were used to analyze sphingolipid concentration by LC–MS/MS [see “Sphingolipid concentration determination by liquid chromatography–tandem mass spectrometry (LC–MS/MS)”]. 180 μL sample was purified by PD SpinTrap G-25 columns (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s protocol to separate saccharose from proteins for Western blot analysis. See “Protein concentration determination by Western blot analysis” for protein concentration analysis of the fractions.

Cholesterol assay of glycosphingolipid-enriched microdomains (GEMs)

“Isolation of glycosphingolipid-enriched microdomains (GEMs)” described samples were also used for the determination of cholesterol in the different fractions. 10 µL standards dissolved in 100% ethanol and 10 µL samples were added to 5 μL of emulsifier solution (15% Brij in 5 mM Tris/HCl, pH 7.5) and incubated in a 96 well plate (Greiner BIO-ONE, Frickenhausen) at room temperature for 15 min. Afterwards 200 μL of the reaction solution was added and incubated for 60 min at 37 °C. The reaction solution was freshly prepared at a ratio of 1:1:0.01 of solution A (0.02 M phenol, 1.84 M MeOH, 1% methyl-β-cyclodextrin, ad 50 mM Tris/HCl pH 7.4), solution B (0.0019 M 4-aminoantipyrin, 1.84 M MeOH, 0.4% Brij (30% w/v), and 50 mM Tris/HCl, pH 7.4) and enzyme solution. The enzyme solution consisted of cholesterol oxidase (12 U/mL) and peroxidase (8 U/mL). Finally, the absorbance at 540 nm was measured by Tecan SpectraFluor Plus Reader (Tecan, Crailsheim, Germany).

Detection of intracellular-induced signaling pathways

For analysis of signaling pathways in MCF-7 cells, the Pathscan^® Intracellular Signaling Array Kit (Cell Signaling, Cambridge, UK) was used. This antibody array detects several phosphorylated signaling proteins. The assay was performed according to the manufacturer’s protocol.

Statistics

Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed with GraphPad Prism 6 software. Significance of means was examined by the indicated statistical test in the legend of the appropriate figure.

Electronic supplementary material

Below is the link to the electronic supplementary material.