Abstract

We have recently established and characterized cellular clones deriving from MDA-MB-231 breast cancer cells that express the human GD3 synthase (GD3S), the enzyme that controls the biosynthesis of b- and c-series gangliosides. The GD3S positive clones show a proliferative phenotype in the absence of serum or growth factors and an increased tumor growth in severe immunodeficient mice. This phenotype results from the constitutive activation of the receptor tyrosine kinase c-Met in spite of the absence of ligand and subsequent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/Akt pathways. Here, we show by mass spectrometry analysis of total glycosphingolipids that GD3 and GD2 are the main gangliosides expressed by the GD3S positive clones. Moreover, GD2 colocalized with c-Met at the plasma membrane and small interfering RNA silencing of the GM2/GD2 synthase efficiently reduced the expression of GD2 as well as c-Met phosphorylation and reversed the proliferative phenotype. Competition assays using anti-GD2 monoclonal antibodies also inhibit proliferation and c-Met phosphorylation of GD3S positive clones in serum-free conditions. Altogether, these results demonstrate the involvement of the disialoganglioside GD2 in MDA-MB-231 cell proliferation via the constitutive activation of c-Met. The accumulation of GD2 in c-Met expressing cells could therefore reinforce the tumorigenicity and aggressiveness of breast cancer tumors.

Introduction

Gangliosides are glycosphingolipids (GSLs) carrying one or several sialic acid residues. They are essentially located on the outer leaflet of the plasma membrane in microdomains named “glycosynapses”, where they can interact with transmembrane receptors or signal transducers involved in cell proliferation and signaling (Hakomori 2002; Todeschini and Hakomori 2008).

GSLs from ganglio-series are classified into four series according to the presence of 0–3 sialic acid residues linked to lactosylceramide (LacCer). The transfer of sialic acid is catalyzed in the Golgi apparatus by specific sialyltransferases [GM3 synthase (ST3Gal V), GD3 synthase (GD3S) and GT3 synthase (ST8Sia V), respectively] that show high specificity toward their glycolipid substrates (Zeng and Yu 2008). LacCer, GM3, GD3 and GT3 are therefore the precursors for 0-, a-, b- and c-series gangliosides (Figure 1), and the biosynthesis of these compounds determines the relative proportion of gangliosides in each series (Figure 1). Afterwards, further monosaccharides, including N-acetylgalactosamine (GalNAc), galactose (Gal) and sialic acid (N-acetylneuraminic acid, Neu5Ac), can be transferred in a stepwise manner by other specific glycosyltransferases (Tettamanti 2004). The steady state level of membrane-associated gangliosides is therefore dependent on the activity of several glycosyltransferases, including ST3Gal V, ST8Sia I (GD3S), ST8Sia V or the β4GalNAc T1 (GM2/GD2 synthase).

Fig. 1.

Biosynthesis of gangliosides. The action of ST3Gal V (GM3 synthase), ST8Sia I (GD3 synthase) and ST8Sia V (GT3 synthase) leads to the biosynthesis of the precursors of a-, b- and c-series gangliosides, respectively. The 0-series gangliosides are directly synthesized from LacCer. Elongation is performed by the sequential action of N-acetylgalactosaminyltransferase (β4GalNAc T1), galactosyltransferase (β3Gal T4) and sialyltransferases (ST3Gal I, ST3Gal II and ST8Sia V). Cer, ceramide; blue circle, Glc; yellow circle, Gal; yellow square, GalNAc; pink diamond, Neu5Ac.

Fig. 1.

Biosynthesis of gangliosides. The action of ST3Gal V (GM3 synthase), ST8Sia I (GD3 synthase) and ST8Sia V (GT3 synthase) leads to the biosynthesis of the precursors of a-, b- and c-series gangliosides, respectively. The 0-series gangliosides are directly synthesized from LacCer. Elongation is performed by the sequential action of N-acetylgalactosaminyltransferase (β4GalNAc T1), galactosyltransferase (β3Gal T4) and sialyltransferases (ST3Gal I, ST3Gal II and ST8Sia V). Cer, ceramide; blue circle, Glc; yellow circle, Gal; yellow square, GalNAc; pink diamond, Neu5Ac.

Normal human tissues mainly express a-series gangliosides (Figure 1), whereas complex gangliosides from b- and c-series are essentially found in developing tissues, during embryogenesis, and mainly restricted to the nervous system in healthy adults (Yamashita et al. 1999). In mammals, the expression of b- and c-series gangliosides increases in pathological conditions including atherosclerosis, neurodegenerative disorders and cancer (Prokazova and Bergelson 1994; Birklé et al. 2003; Ariga et al. 2008). In this context, GD3 and GD2 have been revealed as tumor-associated carbohydrate antigens in neuroectoderm-derived tumors such as melanoma, neuroblastoma and glioblastoma (Furukawa et al. 2006).

It has been clearly demonstrated that complex gangliosides play a key role in tumor growth and metastasis, by mediating cell proliferation, migration, adhesion and angiogenesis (Birklé et al. 2003). Complex gangliosides have also been used as target molecules for cancer immunotherapy, such as GD3 in melanoma (Chapman et al. 2004; Scott et al. 2005; Lo et al. 2010) or GD2 in neuroblastoma (Navid et al. 2010; Yu et al. 2010). However, the molecular functions of individual gangliosides in tumor progression and aggressiveness remain elusive. One mechanism by which gangliosides may exert their effects on proliferation is through the modulation of tyrosine kinase receptor (RTK) activation. For example, GD3S expression in rat pheochromocytoma PC12 cells leads to the cell surface accumulation of GD1b and GT1b, resulting in continuous neurotrophin receptor TrkA activation, enhancing cell growth without nerve growth factor binding (Fukumoto et al. 2000). In A431 human epidermoid carcinoma cells, the activation of epithelial growth factor RTK (EGFR) is inhibited by GM3 through direct carbohydrate–carbohydrate interactions between GM3 and terminal GlcNAc residue on EGFR (Yoon et al. 2006; Kawashima et al. 2009).

In normal breast tissues, complex gangliosides are absent or expressed at very low level, but GD3, 9-O-acetyl-GD3 and 9-O-acetyl-GT3 are oncofetal markers in invasive ductal breast carcinoma (Marquina et al. 1996). Clinical studies have also shown that ST8SIA1, the gene encoding the GD3S, the key enzyme that controls b- and c-series gangliosides biosynthesis, displayed higher expression among estrogen receptor (ER) negative breast cancer tumors (Ruckhäberle et al. 2008). Furthermore, ST8SIA1 expression was associated with a higher histological grade in ER negative tumors (Ruckhäberle et al. 2009).

We have recently developed a cellular model deriving from the triple negative (ER−, PR− and Her2−) MDA-MB-231 breast cancer cell line, expressing the human GD3S and showing a proliferative phenotype in the absence of serum or exogenous growth factors (Cazet et al. 2009). The proliferative capacities of MDA-MB-231 GD3S+ clones in serum-free conditions directly proceed from the constitutive activation of the c-Met receptor (Peschard and Park 2007) and downstream mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide-3 kinase (PI3K)/Akt signaling pathways (Cazet et al. 2010). Moreover, GD3S expression not only promotes cell growth in vitro but also stimulates primary tumor growth in severe combined immunodeficiency mice. Finally, micro-array analysis has shown a higher expression of ST8SIA1 and MET in the basal-like subtype (Sorlie et al. 2003) of human breast tumors (Cazet et al. 2010).

In the present study, we show that GD3 and GD2 are the main gangliosides expressed by MDA-MB-231 GD3S+ clones, whereas control MDA-MB-231 cells accumulate mainly GM3 and GM2. We also demonstrate that GD2 is directly involved in the constitutive activation of c-Met in the absence of the ligand hepatocyte growth factor (HGF)/scatter factor, probably through specific interaction between the c-Met receptor and the oligosaccharide moiety of GD2.

Results

Ganglioside contents of GD3S+ MDA-MB-231 cells

To precisely establish the ganglioside profile of GD3S+ MDA-MB-231 cells, we analyzed the composition of GSL by MALDI-TOF mass spectrometry after permethylation (Figure 2, Table I). Two ceramide isoforms are commonly expressed in human tissues due to the substitution of the sphingosine moiety by palmitic acid C16:0 or lignoceric acid C24:0 (Figure 2, Table I). As expected, wild-type or empty vector-transfected MDA-MB-231 (control) cells only expressed a-series gangliosides, mainly GM3 and GM2 (Figure 2A and B). HPTLC analysis confirmed that wild-type MDA-MB-231 cells only expressed a-series gangliosides, mainly GM3 and GM2, GM1 being expressed at a low level (Figure 3, lanes A5 and B5). Glucosylceramide and LacCer were also detected, as well as globo-series GSLs Gb3 and Gb4. On the other hand, both GD3S+ clones (clones 4 and 11) accumulated b- and c-series gangliosides, as it was previously shown by flow cytometry and confocal microscopy using different anti-ganglioside mAbs (Cazet et al. 2009). GD3 and GD2 represent the most predominant complex gangliosides, whereas lower amount of GD1b, GT3 and GT2 were detected (Figure 2C and D). These results fit well with the high expression of the β4GalNAc T1 in MDA-MB-231 cells, as previously shown by QPCR (Cazet et al. 2009). In parallel, GM3 and GM2 expression levels were reduced in MDA-MB-231 GD3S+ cells compared with control cells. Similarly, HPTLC analysis confirmed that both GD3S+ clones mainly expressed GD3 and GD2 (Figure 3, lanes A4 and B4). Moreover, immunostaining using the A2B5 mAb allows also to detect GT3 and GT2 in GD3S+ clones, which cannot be distinguished from GD1b and GT1b on the resorcinol stained HPTLC plate (Figure 3, lane B6). The structures of individual GSLs were confirmed by mass spectrometry fragmentation analyses by MALDI-TOF/TOF (data not shown). Finally, we were able to show by immunocytochemistry and confocal microscopy the colocalization of GD2 and c-Met at the plasma membrane of GD3S+ clones (Figure 4).

Table I.

Compositional assignments of singly charged sodiated molecular ions [M + Na]+, observed in MALDI-TOF mass spectrometry spectra of permethylated glycolipids from MDA-MB-231 GD3S+ cells

Fatty acids Glycolipids Calculated mono-isotopic molecular masses Wild type Control Clone 4 Clone 11 
16:0 GlcCer 806.6487 806.3968 806.5892 806.6505 806.6308 
24:0 GlcCer 918.7745 918.4902 918.7043 918.7890 918.7596 
16:0 LacCer 1010.7485 1010.4339 1010.6812 1010.7537 1010.7374 
24:0 LacCer 1122.8743 1122.5191 1122.8012 1122.8852 1122.8603 
16:0 Gb3 1214.8483 1214.4646 1214.7607 1214.8531 1214.8289 
24:0 Gb3 1326.9741 1326.5504 1326.8753 1326.9801 1326.9451 
16:0 GM3 1371.9222 1371.4816 1371.8200 1371.94.5 1371.9189 
24:0 GM3 1484.0480 1483.5651 1483.9274 1484.0325 1484.0151 
16:0 Gb4 1459.9746 1459.5238 1459.8734 1459.9753 1459.9583 
24:0 Gb4 1572.1004 1571.6016 1571.9842 1572.0918 1572.0716 
16:0 GM2 1617.0485 1616.5302 1616.9290 1617.0441 1617.0287 
24:0 GM2 1729.1743 1728.6238 1729.0503 1729.1538 1729.1384 
16:0 GD3 1733.0958   1733.0929 1733.0783 
24:0 GD3 1845.2216   1845.1859 1845.1750 
16:0 GM1a/b 1821.1483 1820.5616 (GM1a) 1821.0126 (GM1a) 1821.1569 (GM1b) 1821.1408 (GM1b) 
24:0 GM1a/b 1933.2741 1932.6440 (GM1a) 1933.1304 (GM1a) 1933.2096 (GM1b) 1933.2078 (GM1b) 
16:0 GD2 1978.2222   1978.2035 1978.1653 
24:0 GD2 2090.3480   2090.3065 2090.2845 
16:0 GT3 2094.2695   2294.2622 2294.2524 
24:0 GT3 2206.3953   2206.3362 2206.3191 
16:0 GD1a/b 2182.3219 2182.6238 (GD1a) 2183.1728 (GD1a) 2182.2870 (GD1b) 2182.2808 (GD1b) 
24:0 GD1a/b 2294.4477 2293.6881 (GD1a) 2294.2640 (GD1a) 2294.3905 (GD1b) 2294.3722 (GD1b) 
16:0 GT2 2339.3958   2340.3806 2340.3997 
24:0 GT2 2451.5216   2451.4548 2452.4352 
Fatty acids Glycolipids Calculated mono-isotopic molecular masses Wild type Control Clone 4 Clone 11 
16:0 GlcCer 806.6487 806.3968 806.5892 806.6505 806.6308 
24:0 GlcCer 918.7745 918.4902 918.7043 918.7890 918.7596 
16:0 LacCer 1010.7485 1010.4339 1010.6812 1010.7537 1010.7374 
24:0 LacCer 1122.8743 1122.5191 1122.8012 1122.8852 1122.8603 
16:0 Gb3 1214.8483 1214.4646 1214.7607 1214.8531 1214.8289 
24:0 Gb3 1326.9741 1326.5504 1326.8753 1326.9801 1326.9451 
16:0 GM3 1371.9222 1371.4816 1371.8200 1371.94.5 1371.9189 
24:0 GM3 1484.0480 1483.5651 1483.9274 1484.0325 1484.0151 
16:0 Gb4 1459.9746 1459.5238 1459.8734 1459.9753 1459.9583 
24:0 Gb4 1572.1004 1571.6016 1571.9842 1572.0918 1572.0716 
16:0 GM2 1617.0485 1616.5302 1616.9290 1617.0441 1617.0287 
24:0 GM2 1729.1743 1728.6238 1729.0503 1729.1538 1729.1384 
16:0 GD3 1733.0958   1733.0929 1733.0783 
24:0 GD3 1845.2216   1845.1859 1845.1750 
16:0 GM1a/b 1821.1483 1820.5616 (GM1a) 1821.0126 (GM1a) 1821.1569 (GM1b) 1821.1408 (GM1b) 
24:0 GM1a/b 1933.2741 1932.6440 (GM1a) 1933.1304 (GM1a) 1933.2096 (GM1b) 1933.2078 (GM1b) 
16:0 GD2 1978.2222   1978.2035 1978.1653 
24:0 GD2 2090.3480   2090.3065 2090.2845 
16:0 GT3 2094.2695   2294.2622 2294.2524 
24:0 GT3 2206.3953   2206.3362 2206.3191 
16:0 GD1a/b 2182.3219 2182.6238 (GD1a) 2183.1728 (GD1a) 2182.2870 (GD1b) 2182.2808 (GD1b) 
24:0 GD1a/b 2294.4477 2293.6881 (GD1a) 2294.2640 (GD1a) 2294.3905 (GD1b) 2294.3722 (GD1b) 
16:0 GT2 2339.3958   2340.3806 2340.3997 
24:0 GT2 2451.5216   2451.4548 2452.4352 

Only masses corresponding to N-palmitoyl- (C16:0) or N-lignoceroyl- (C24:0) 2-amino-4-octadecene-1,3-diol (sphingosine) are indicated. Assignments were confirmed by mass spectrometry fragmentation of permethylated glycolipids by MALDI-TOF/TOF (data not shown). WT, non-transfected cells; control, cells transfected with empty vector; clones 4 and 11, cells transfected with human GD3S cDNA; LacCer, lactosylceramide; GlcCer, glucosylceramide; Gb, globoside.

Fig. 2.

MALDI-TOF analysis of permethylated GSLs isolated from MDA-MB-231 GD3S+ cells. Permethylated GSLs isolated from (A) wild-type MDA-MB-231, (B) control MDA-MB-231 transfected with empty vector, (C) GD3S+ MDA-MB-231 clone 4 and (D) clone 11 were analyzed by MALDI-TOF mass spectrometry. Gangliosides from b- or c-series were not detected in wild-type and control cells, whereas complex gangliosides containing 2–3 sialic acid residues (i.e. GD3, GD2, GT3, GT2) were detected in MDA-MB-231 GD3S+ cells. Black circle, Glc; gray circle, Gal; gray square, GalNAc; dark gray diamond, Neu5Ac; graphic, Ceramide either N-palmitoyl-sphingosine or N-lignoceryl-sphingosine.

Fig. 2.

MALDI-TOF analysis of permethylated GSLs isolated from MDA-MB-231 GD3S+ cells. Permethylated GSLs isolated from (A) wild-type MDA-MB-231, (B) control MDA-MB-231 transfected with empty vector, (C) GD3S+ MDA-MB-231 clone 4 and (D) clone 11 were analyzed by MALDI-TOF mass spectrometry. Gangliosides from b- or c-series were not detected in wild-type and control cells, whereas complex gangliosides containing 2–3 sialic acid residues (i.e. GD3, GD2, GT3, GT2) were detected in MDA-MB-231 GD3S+ cells. Black circle, Glc; gray circle, Gal; gray square, GalNAc; dark gray diamond, Neu5Ac; graphic, Ceramide either N-palmitoyl-sphingosine or N-lignoceryl-sphingosine.

Fig. 3.

HPTLC analysis of gangliosides isolated from MDA-MB-231 GD3S+ cells. (A) HPTLC analysis of gangliosides from wild-type and GD3S+ MDA-MB-231 clone 4. Gangliosides were separated onto HPTLC glass plates in CHCl3/CH3OH/0.2% CaCl2 (55:45:10, v/v/v) and visualized with a resorcinol/HCl spray reagent 5 min at 150°C. Lane 1, GM3 and GD3 from human melanoma tumors; 2, total bovine brain gangliosides; 3, polysialoganglioside fraction from human melanoma tumors; 4, GD3S+ MDA-MB-231 clone 4; 5, wild-type MDA-MB-231 cells. (B) Immunostaining of GD3S+ MDA-MB-231 clone 11 with the A2B5 mAb. Left panel: visualization of gangliosides with a resorcinol/HCl spray reagent 5 min at 150°C. Right panel: visualization of gangliosides with the A2B5 mAb. Lane 1, GM3 and GD3 from human melanoma tumors; 2, total bovine brain gangliosides; 3, polysialoganglioside fraction from human melanoma tumors; 4 and 6, GD3S+ MDA-MB-231 clone 11; 5 and 7, wild-type MDA-MB-231 cells.

Fig. 3.

HPTLC analysis of gangliosides isolated from MDA-MB-231 GD3S+ cells. (A) HPTLC analysis of gangliosides from wild-type and GD3S+ MDA-MB-231 clone 4. Gangliosides were separated onto HPTLC glass plates in CHCl3/CH3OH/0.2% CaCl2 (55:45:10, v/v/v) and visualized with a resorcinol/HCl spray reagent 5 min at 150°C. Lane 1, GM3 and GD3 from human melanoma tumors; 2, total bovine brain gangliosides; 3, polysialoganglioside fraction from human melanoma tumors; 4, GD3S+ MDA-MB-231 clone 4; 5, wild-type MDA-MB-231 cells. (B) Immunostaining of GD3S+ MDA-MB-231 clone 11 with the A2B5 mAb. Left panel: visualization of gangliosides with a resorcinol/HCl spray reagent 5 min at 150°C. Right panel: visualization of gangliosides with the A2B5 mAb. Lane 1, GM3 and GD3 from human melanoma tumors; 2, total bovine brain gangliosides; 3, polysialoganglioside fraction from human melanoma tumors; 4 and 6, GD3S+ MDA-MB-231 clone 11; 5 and 7, wild-type MDA-MB-231 cells.

Fig. 4.

Colocalization of GD2 and c-Met at the plasma membrane of GD3S+ clones. Control and GD3S+ MDA-MB-231 cells were incubated with goat AF276 anti-c-Met and mouse S220-51 anti-GD2 mAbs and revealed with red-fluorescent Alexa Fluor® 647 conjugated anti-goat IgG and green-fluorescent FITC-conjugated anti-mouse IgG. The nuclei are counterstained with Hoechst 33258. Slides were observed with a LSM 710 Laser Scanning Microscope (Carl Zeiss), numerical aperture PLAN-APOCHROMAT 63× NA 1.4 with the ZEN acquisition software.

Fig. 4.

Colocalization of GD2 and c-Met at the plasma membrane of GD3S+ clones. Control and GD3S+ MDA-MB-231 cells were incubated with goat AF276 anti-c-Met and mouse S220-51 anti-GD2 mAbs and revealed with red-fluorescent Alexa Fluor® 647 conjugated anti-goat IgG and green-fluorescent FITC-conjugated anti-mouse IgG. The nuclei are counterstained with Hoechst 33258. Slides were observed with a LSM 710 Laser Scanning Microscope (Carl Zeiss), numerical aperture PLAN-APOCHROMAT 63× NA 1.4 with the ZEN acquisition software.

SiRNA inhibition of β4GalNAc T1 expression strongly reduces c-Met activation and reverses the proliferative phenotype of GD3S+ cells

In order to determine the role of GD2 in the activation of c-Met and proliferative phenotype, the GM2/GD2 synthase (β4GalNAc T1) expression was inhibited in GD3S+ cells with siRNA sequences. The ability of the siRNA to specifically silence the B4GALNACT1 gene was evaluated by QPCR and flow cytometry analysis. In comparison with control siRNA, the silencing of the GM2/GD2 synthase resulted in 90, 87 and 90% decreases of relative β4GalNAc T1 mRNA expression in MDA-MB-231 control, clone 4 and clone 11, respectively (Figure 5A). In parallel, siRNA efficiently reduced the expression of GD2 in the both clones, whereas GD3 and GT3 expression were increased (Figure 5B). As shown in Figure 6A, silencing of GM2/GD2 synthase decreased proliferation of both GD3S+ clones during the first 3 days of culture, demonstrating the involvement of GalNAc-substituted complex gangliosides, mainly GD2, in GD3S+ cells proliferation in deprivation conditions. The phosphorylation status of 42 RTKs was simultaneously examined in MDA-MB-231 GD3S+ cells 24 h after transfection with corresponding siRNA using phospho-RTK arrays. As previously described (Cazet et al. 2010), GD3S+ MDA-MB-231 cells displayed a strong phosphorylation of the c-Met receptor (coordinates c3, c4) in control conditions (Figure 6B). The phosphorylation of EGFR (coordinates d1, d2) and of the receptor for angiopoietins Tie-2 (coordinates b1, b2) is also observed. In parallel, inhibition of GM2/GD2 synthase expression strongly decreased the c-Met phosphorylation compared with MDA-MB-231 GD3S+ cells transfected with control siRNA. A similar decrease in Tie-2 (coordinates b1, b2) and a slight decrease in EGFR (coordinates d1, d2) phosphorylation were also observed (Figure 6B). Even if siRNA treatment strongly inhibited β4GalNAc T1 mRNA expression (Figure 5A), a slight activation of c-Met remained detectable on phospho-RTK arrays (Figure 6B). This could explain why the growth rate of siRNA GD3S+-treated cells is not completely reduced to the control level.

Fig. 5.

SiRNA inhibition of β4GalNAc T1 strongly reduces GD2 expression in GD3S+ cells. (A) SiRNA inhibition of β4GalNAc T1. Control and GD3S+ clones were transfected with siRNA-targeting B4GALNACT1 or with a scramble sequence. Total RNA was reverse-transcribed and proceeded for QPCR. Relative quantification of B4GALNACT1 expression was performed by the method described by Livak and Schmittgen (2001) and normalized to HPRT. Grey bars, control siRNA; open bars, B4GALNACT1 siRNA. (B) Inhibition of GD2 expression. Control and GD3S+ clones were transfected with siRNA-targeting B4GALNACT1 or with a scramble sequence. Cells were harvested and incubated with anti-GD3 R24, anti-GD2 S220-51 or anti-GT3 A2B5 mAbs. Grey peaks, control siRNA; open peaks, B4GALNACT1 siRNA.

Fig. 5.

SiRNA inhibition of β4GalNAc T1 strongly reduces GD2 expression in GD3S+ cells. (A) SiRNA inhibition of β4GalNAc T1. Control and GD3S+ clones were transfected with siRNA-targeting B4GALNACT1 or with a scramble sequence. Total RNA was reverse-transcribed and proceeded for QPCR. Relative quantification of B4GALNACT1 expression was performed by the method described by Livak and Schmittgen (2001) and normalized to HPRT. Grey bars, control siRNA; open bars, B4GALNACT1 siRNA. (B) Inhibition of GD2 expression. Control and GD3S+ clones were transfected with siRNA-targeting B4GALNACT1 or with a scramble sequence. Cells were harvested and incubated with anti-GD3 R24, anti-GD2 S220-51 or anti-GT3 A2B5 mAbs. Grey peaks, control siRNA; open peaks, B4GALNACT1 siRNA.

Fig. 6.

SiRNA inhibition of β4GalNAc T1 strongly reduces c-Met activation and reverses the proliferative phenotype of GD3S+ cells. (A) Inhibition of GD2 expression reduces GD3S+ clones proliferation in serum-free conditions. One day after transfection, cells were seeded in 96-well plates and cultured during 5 days in serum-free medium as described in Materials and Methods section. Cell growth was analyzed by MTS assay. Counting was performed in 16 wells and data are the mean of three independent manipulations. **P < 0.01, GD3S+ vs. control. (B) Phospho-array analysis. Total cell lysates from control, GD3S+ clone 4 and clone 11, transfected with specific siRNA-targeting B4GALNACT1 or with a control sequence, were subjected to phospho-RTK array. Phospho-RTK array coordinates are given on the left side of the figure. Black dots represent Phospho-Tyrosine positive control; a1, a2: EphA6; a3, a4: EphA7; a5, a6: EphB1; a7, a8: EphB2; a9, a10: EphB4; a11, a12: EphB6; a13, a14: Mouse IgG1 negative control; a15, a16: Mouse IgG2A negative control; a17, a18: Mouse IgG2B negative control; a19, a20: Goat IgG negative control; a21, a22: PBS negative control; b1, b2: Tie-2; b3, b4: TrkA; b5, b6: TrkB; b7, b8: TrkC; b9, b10: VEGFR1; b11, b12: VEGFR2; b13, b14: VEGFR3; b15, b16: MuSK; b17, b18: EphA1; b19, b20: EphA2; b21, b22: EphA3; b23, b24: EphA4; c1, c2: Mer; c3, c4: c-Met; c5, c6: MSPR; c7, c8: PDGFRα; c9, c10: PDGFRβ; c11, c12: SCFR; c13, c14: Flt-3; c15, c16: M-CSFR; c17, c18: c-Ret; c19, c20: ROR1; c21, c22: ROR2; c23, c24: Tie-1; d1, d2: EGFR; d3, d4: ErbB2; d5, d6: ErbB3; d7, d8: ErbB4; d9, d10: FGFR1; d11, d12: FGFR2α; d13, d14: FGFR3; d15, d16: FGFR4; d17, d18: Insulin R; d19, d20: IGF-I R; d21, d22: Axl; d23, d24: Dtk.

Fig. 6.

SiRNA inhibition of β4GalNAc T1 strongly reduces c-Met activation and reverses the proliferative phenotype of GD3S+ cells. (A) Inhibition of GD2 expression reduces GD3S+ clones proliferation in serum-free conditions. One day after transfection, cells were seeded in 96-well plates and cultured during 5 days in serum-free medium as described in Materials and Methods section. Cell growth was analyzed by MTS assay. Counting was performed in 16 wells and data are the mean of three independent manipulations. **P < 0.01, GD3S+ vs. control. (B) Phospho-array analysis. Total cell lysates from control, GD3S+ clone 4 and clone 11, transfected with specific siRNA-targeting B4GALNACT1 or with a control sequence, were subjected to phospho-RTK array. Phospho-RTK array coordinates are given on the left side of the figure. Black dots represent Phospho-Tyrosine positive control; a1, a2: EphA6; a3, a4: EphA7; a5, a6: EphB1; a7, a8: EphB2; a9, a10: EphB4; a11, a12: EphB6; a13, a14: Mouse IgG1 negative control; a15, a16: Mouse IgG2A negative control; a17, a18: Mouse IgG2B negative control; a19, a20: Goat IgG negative control; a21, a22: PBS negative control; b1, b2: Tie-2; b3, b4: TrkA; b5, b6: TrkB; b7, b8: TrkC; b9, b10: VEGFR1; b11, b12: VEGFR2; b13, b14: VEGFR3; b15, b16: MuSK; b17, b18: EphA1; b19, b20: EphA2; b21, b22: EphA3; b23, b24: EphA4; c1, c2: Mer; c3, c4: c-Met; c5, c6: MSPR; c7, c8: PDGFRα; c9, c10: PDGFRβ; c11, c12: SCFR; c13, c14: Flt-3; c15, c16: M-CSFR; c17, c18: c-Ret; c19, c20: ROR1; c21, c22: ROR2; c23, c24: Tie-1; d1, d2: EGFR; d3, d4: ErbB2; d5, d6: ErbB3; d7, d8: ErbB4; d9, d10: FGFR1; d11, d12: FGFR2α; d13, d14: FGFR3; d15, d16: FGFR4; d17, d18: Insulin R; d19, d20: IGF-I R; d21, d22: Axl; d23, d24: Dtk.

Inhibition of cell growth and c-Met activation by the anti-GD2 mAb

Effect of the anti-GD2 mAb on cell proliferation was then analyzed by adding affinity purified specific mAbs to the culture medium. The high proliferative capacity of the MDA-MB-231 GD3S+ was strongly decreased in the presence of the anti-GD2 4G2 mAb, whereas the anti-GD3 4F6 mAb has no significant effect on cell proliferation. The inhibition effect of anti-GD2 was dependent on the mAb concentration and became significant even at 15 µg/mL (Figure 7A). In parallel, no morphological change was observed in GD3S+ clones after anti-GD2 mAb treatment (data not shown) and the anti-GD2 mAb has no effect on the cellular level of GD2 (Supplementary data). Finally, as shown in Figure 7B, the anti-GD2 mAb also strongly decreased the phosphorylation of c-Met, whereas anti-GD3 had no effect.

Fig. 7.

Inhibition of the cell growth and c-Met activation of MDA-MB-231 GD3S+ by the anti-GD2 mAb. (A) Cell proliferation of GD3S+ clones treated with 15 µg/mL of the anti-GD2 4G2 mAb in serum-free conditions was determined by MTS assay after 4 days of culture. Each measure was performed in eight wells and data are the mean of three independent manipulations. *P < 0.05. (B) Analysis of c-Met phosphorylation level after addition of the anti-GD2 mAb. MDA-MB-231 control and GD3S+ clones were treated for 6 h with 30 µg/mL of the 4G2 mAb and then used for an immunoblotting with a specific anti-phospho-Met or an anti-Met antibody as described in Materials and methods section. The anti-GD3 4F6 mAb and an irrelevant mAb were used as a control.

Fig. 7.

Inhibition of the cell growth and c-Met activation of MDA-MB-231 GD3S+ by the anti-GD2 mAb. (A) Cell proliferation of GD3S+ clones treated with 15 µg/mL of the anti-GD2 4G2 mAb in serum-free conditions was determined by MTS assay after 4 days of culture. Each measure was performed in eight wells and data are the mean of three independent manipulations. *P < 0.05. (B) Analysis of c-Met phosphorylation level after addition of the anti-GD2 mAb. MDA-MB-231 control and GD3S+ clones were treated for 6 h with 30 µg/mL of the 4G2 mAb and then used for an immunoblotting with a specific anti-phospho-Met or an anti-Met antibody as described in Materials and methods section. The anti-GD3 4F6 mAb and an irrelevant mAb were used as a control.

Discussion

The pharmacological and genetic control of gangliosides expression has been applied during the past years to study the influence of b-series gangliosides on cell behavior, including cell proliferation, and accumulating evidences indicate the tumor-specific and malignant phenotype-associated expression of GD2. For example, GD2 expression is required and sufficient to drive human small cell lung cancer (SCLC) proliferation, migration and invasion in vitro as well as tumor growth (Ko et al. 2006). In this context, the disialoganglioside GD2 represents a valid therapeutic target for cancer immunotherapy (Ragupathi et al. 2003; Yvon et al. 2009; Lo et al. 2010).

Growth factor receptors are characterized by a common susceptibility to gangliosides, which modulate the receptor-associated tyrosine kinase activity in glycosynaptic microdomains (Miljan and Bremer 2002; Kaucic et al. 2006). Studies conducted over the past decade have demonstrated that a-series gangliosides inhibit c-Met signaling. GD1a inhibits HGF-induced motility of cancer cells through suppression of tyrosine phosphorylation of c-Met (Hyuga et al. 2001). GM3 and GM2 can form heterodimers that specifically interact with tetraspanin family member CD82, and GM3/GM2/CD82 complexes inhibit c-Met activation and cross-talk with integrins, providing a basis for the control of cell proliferation and invasiveness (Todeschini et al. 2007, 2008). In parallel, we have demonstrated that the expression of GD3S and complex gangliosides in MDA-MB-231 cells lead to a proliferative phenotype by a positive regulation of c-Met phosphorylation and subsequent signal transduction MEK/ERK and PI3K/Akt pathways (Cazet et al. 2010). Moreover, the proliferative phenotype of GD3S+ cells was due to a HGF-independent activation of c-Met. Thus, c-Met is activated in the absence of HGF and 5D5 Fab, which inhibits HGF-Met association and ligand-dependent activation, does not affect Met activation (Cazet et al. 2010). The reduction in a-series ganglioside levels cannot therefore explained c-Met activation, indicating that the HGF-independent activation of c-Met in GD3S+ MDA-MB-231 cells is induced by b-series gangliosides. Consequently, these studies seem to indicate that the ganglioside-dependent regulation of c-Met is related to ganglioside sialylation status, a-series inhibiting c-Met phosphorylation, whereas b- or c-series gangliosides strongly activate the receptor in a ligand-independent manner.

Here, we show by mass spectrometry and HPTLC analysis of total GSLs that GD2 is the main ganglioside expressed by MDA-MB-231 GD3S+ cells. SiRNAs silencing of the GM2/GD2 synthase reversed the proliferative phenotype due to the strong decrease in c-Met phosphorylation. Accumulation of b- and c-series ganglioside precursors, GD3 and GT3, respectively, has no effect on c-Met activation. Altogether, these results show that GD2 contributes to the constitutive activation of the c-Met axis, independently on HGF binding, leading to enhanced signaling, as observed in the basal-like subtype of breast cancer (Graveel et al. 2009; Ponzo et al. 2009).

Although the interactions between GD2 and c-Met remain to be demonstrated, the inhibition of cell proliferation by the anti-GD2 mAb clearly indicates the role of the oligosaccharide moiety of GD2 in c-Met activation. GD2-dependent c-Met activation might be achieved by the control of tyrosine kinase activity by a direct binding through carbohydrate–carbohydrate interactions between GD2 and N-glycosylated chains of the receptor, as it has been previously demonstrated for the EGFR (Yoon et al. 2006; Kawashima et al. 2009). On the other hand, the GD2-induced c-Met constitutive activation could be also dependent on a cross-talk between GD2, c-Met and other signaling molecules. Such complexes have been reported to modulate RTK activity (Todeschini et al. 2007, 2008; Park et al. 2009). Cross-communication between integrins and RTKs is thought to be required for maximal activation of the Ras–mitogen-activated protein kinase (MAPK) signal transduction pathway that drives cell proliferation. In parallel, the importance of ganglioside composition in defining integrin function is also well-documented (Cheresh et al. 1986; Wang et al. 2001, 2002). GD2 physically interacts with integrin and focal adhesion kinase (FAK) to activate the downstream signaling pathway MEK/ERK, playing pivotal role on cell proliferation and malignant properties of SCLC (Yoshida et al. 2001; Aixinjueluo et al. 2005). In this context, the proliferative phenotype of MDA-MB-231 GD3S+ cells could be due to the formation of a tertiary complex consisting of GD2, integrins and c-Met, which could contribute to the constitutive trans-phosphorylation of the receptor and reinforce the malignant properties of the cells. Eventual interactions between disialoganglioside GD2 and integrins should be clarified using different approaches, such as co-immunoprecipitation, cross-linking and confocal microscopy, to further understand the mechanism of specific c-Met activation.

Interestingly, incubation with the anti-GD2 mAb seems to induce cell death of MDA-MB-231 GD3S+ cells after 4 days of culture in deprivation conditions (data not shown). Molecular mechanisms inducing the cell death of cancer cells by the anti-GD2 mAb were investigated previously. Treatment of M21 melanoma cells with the anti-GD2 mAb causes cell rounding and detachment from a fibronectin substrate (Cheresh and Klier 1986). Furthermore, GD2-positive lung cancer cells treated with the anti-GD2 mAb undergo anoikis through the conformational changes of integrin molecules, subsequent FAK dephosphorylation and p38/MAPK activation (Aixinjueluo et al. 2005). The mechanism of cell death induction needs to be clarified in MDA-MB-231 GD3S+ cells but should provide new insights toward immunotherapy application in the drug-resistant breast cancer basal-like subtype.

In conclusion, by using a cellular model that mimic the in vivo situation that could occur in the basal-like subtype of breast cancer tumors expressing the GD3S, we have demonstrated that the oligosaccharide moiety of GD2 specifically activates c-Met and subsequent transduction pathways, enhancing the oncogenic effect of the c-Met receptor. This underlies the need to develop such cellular models and sheds light on the effect of ganglioside expression on breast cancer cell signaling.

Material and methods

Antibodies and reagents

Anti-GD3 R24 monoclonal antibody (mAb; Pukel et al. 1982) was purchased from Abcam (Paris, France) and anti-GD2 S220-51 mAb from Seikagaku Corp. (Tokyo, Japan). Anti-GD3 4F6, anti-GD2 4G2 (Portoukalian et al. 1993) and anti-GT3 A2B5 (Dubois et al. 1990) mAbs were kindly provided by Prof. Jacques Portoukalian (Department of Transplantation and Clinical Immunology, Claude Bernard University and Edouard Herriot Hospital, Lyon, France). The 4F6 and 4G2 mAbs were purified from conditioned culture media by affinity chromatography on protein G column (Sigma-Aldrich, Lyon, France), and protein concentration was determined by the Bio-Rad RC protein assay kit II (Bio-Rad, Marnes-la-Coquette, France). Anti-β-actin, mouse mAb against the COOH-terminal region of human Met and rabbit polyclonal Ab against phosphorylated tyrosines 1234 and 1235 of the Met kinase domain were purchased from Cell Signaling Technology (Saint-Quentin-en-Yvelines, France). Goat anti-c-Met affinity purified polyclonal immunoglobulin G (IgG) AF276 was from R&D Systems Europe (Lille, France). Anti-rabbit and anti-mouse IgG conjugated with horseradish peroxidase, and fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse IgG was purchased from GE Healthcare (Templemars, France). FITC-conjugated rabbit anti-mouse IgM and Alexa Fluor® 647 chicken anti-goat IgG were purchased from Molecular Probes (Invitrogen, Cergy-Pontoise, France). FITC-conjugated goat anti-mouse IgG was from Sigma-Aldrich. Small interfering RNAs (siRNAs) were manufactured by Dharmacon (Thermo Scientific, Illkirch, France) and quantitative real-time polymerase chain reaction (QPCR) primers were synthesized by Eurogentec (Seraing, Belgium).

Cell culture

Cell culture reagents were purchased from Lonza (Levallois-Perret, France). The breast cancer cell line MDA-MB-231 was obtained from the American Type Cell Culture Collection (Rockville, MD). Cells were routinely grown in monolayers and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/mL of penicillin–streptomycin, at 37°C in 5% CO2. MDA-MB-231 control (empty vector transfected) and MDA-MB-231 GD3S+ clones 4 and 11 were obtained as previously described (Cazet et al. 2009) and cultured in the presence of 1 mg/mL of G418 (Invitrogen).

Extraction and preparation of glycolipids

Twenty dishes (10 cm diameter) of cultured cells were washed twice with ice-cold phosphate-buffered saline (PBS) and cells were scraped and homogenized. Cells were suspended in 200 µL of water and sonicated on ice. The resulting material was dried under vacuum and sequentially extracted by CHCl3/CH3OH (2:1, v/v), CHCl3/CH3OH (1:1, v/v) and CHCl3/CH3OH/H2O (1:2:0.8, v/v/v). Supernatants were pooled, dried and subjected to a mild saponification in 0.1 M NaOH in CHCl3/CH3OH (1:1) at 37°C for 2 h and then evaporated to dryness (Schnaar 1994). Samples were reconstituted in CH3OH/H2O (1:1, v/v) and applied to a C18 Sep-Pak cartridge (Waters, Milford, MA) equilibrated in the same solvent system. After washing with five volumes of CH3OH/H2O (1:1, v/v), GSLs were eluted by two volumes of CH3OH, two volumes of CHCl3/CH3OH (1:1, v/v) and two volumes of CHCl3/CH3OH (2:1, v/v).

Mass spectrometry analysis of GSLs

Prior to mass spectrometry analysis, GSLs were permethylated according to Ciucanu and Kerek (1984). Briefly, compounds were incubated 2 h in a suspension of 200 mg/mL of NaOH in dry dimethyl sulfoxide (300 µL) and ICH3 (200 µL). The methylated derivatives were extracted in CHCl3 and washed several times with water. The reagents were evaporated and the sample was dissolved in CHCl3 in the appropriate dilution. Mass spectrometry analysis of permethylated GSLs was performed by matrix-assisted laser desorption-ionization (MALDI) time-of-flight (TOF) on a Voyager Elite reflectron mass spectrometer (PerSeptive Biosystems, Framingham, MA), equipped with a 337-nm UV laser. Samples were prepared by mixing on a tube 5 µL of diluted permethylated derivatives solution in CHCl3 and 5 µL of 2,5-dihydroxybenzoic acid matrix solution [10 mg/mL dissolved in CHCl3/CH3OH (1:1, v/v)]. The mixtures (2 µL) were then spotted on the target plate and air-dried.

Thin-layer chromatography analysis of gangliosides

Standard gangliosides were purified from human melanoma tumors (Portoukalian et al. 1979) and separated into two fractions, one with GM3 and GD3 and the second with the more complex gangliosides. Bovine brain gangliosides were from Sigma-Aldrich. The gangliosides were applied onto high-performance thin-layer chromatography (HPTLC) glass plates (WWR, Paris, France) and migrated in CHCl3/CH3OH/0.2% CaCl2 (55:45:10, v/v/v). The gangliosides spots were visualized with the resorcinol/HCl spray reagent (Svennerholm 1963) 5 min at 150°C. Immunostaining was carried out using an A2B5 mAb to detect polysialogangliosides (Dubois et al. 1990). Standards and gangliosides of clone 11 were migrated on an aluminum-backed HPTLC plate in the same solvent system as above and processed as described (Portoukalian and Bouchon 1986).

Immunofluorescence staining

MDA-MB-231 cells were plated on glass coverslips in 6-well plates (1.6 × 104 cells/well). The next day, cells were washed and fixed 30 min in 4% paraformaldehyde at room temperature. After washing, cells were blocked in PBS/5% bovine serum albumin (BSA; Sigma-Aldrich) for 1 h. Primary antibodies (goat anti-c-Met AF276, mouse anti-GD2 S220-51 or both) were incubated for 1 h. The cells were washed with PBS and incubated 1 h with a combination of secondary antibodies (red-fluorescent Alexa Fluor® 647-conjugated anti-goat IgG and green-fluorescent FITC-conjugated anti-mouse IgG). Cells were then washed with PBS and the nuclei counterstained with Hoechst 33258. Coverslips were mounted with Glycergel mounting medium (Dako, Carpenteria, CA). Fluorescence was examined in oil immersion at 21°C. For confocal fluorescence microscopy, slides were observed in an LSM 710 Laser Scanning Microscope (Carl Zeiss, Thornwood, NY), numerical aperture PLAN-APOCHROMAT 63× NA 1.4 with the ZEN acquisition software.

RNA interference assays

Cells were transfected with 200 nM duplexes siRNA-targeting B4GALNACT1 (L-011279-00) or a scramble sequence using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's instructions with slight modifications. Cells were transfected in OptiMEM medium, in the absence of FBS. Two cycles of siRNA were done to achieve maximal knockdown. Cells were transfected twice with corresponding siRNAs at t = 0 h and t = 48 h and were collected 24 h later for QPCR, cell growth and phospho-array assays, and 48 h later for flow cytometry analysis.

Reverse transcription and QPCR

Total RNA was extracted using the Nucleospin RNA II (Macherey-Nagel, Hoerdt, France) according to the protocol provided by the manufacturer. Total RNA (1 µg) was reverse transcribed using first-strand cDNA synthesis kit (Amersham Biosciences, Freiburg, Germany). QPCR and subsequent analysis were performed using the Mx-3005P Quantitative System (Stratagene, Amsterdam, the Netherlands). Primer pairs for B4GALNACT1 and HPRT (hypoxanthine phosphoribosyltransferase) transcripts were described previously (Cazet et al. 2009). QPCRs (25 µL) were performed using 2× SYBR® Green Universal QPCR Master Mix (Stratagene) with 2 µL of 1:5 cDNA dilution and 300 nM final concentration of each primer. QPCR conditions were as follows: 95°C for 30 s, 51°C for 45 s, 72°C for 30 s (40 cycles). Quantification was performed by the method described by Livak and Schmittgen (2001). Serial dilutions of the appropriate positive control cDNA sample were used to create standard curves for relative quantification and B4GALNACT1 transcripts were normalized to HPRT expression. Assays were performed in triplicate and QPCR amplification was repeated three times. Negative control reactions were performed by replacing cDNA templates by sterile water or corresponding total RNA samples.

Flow cytometry analysis

Cells were detached by 4 mM ethylenediaminetetraacetic acid (EDTA) in PBS and incubated for 1 h at 4°C with anti-gangliosides mAbs: anti-GD3 R24 (1:100), anti-GD2 S220-51 (1:75) and anti-GT3 A2B5 (1:10), diluted in PBS containing 0.5% BSA. After washing with PBS/0.5% BSA, cells were incubated for 1 h on ice with FITC-conjugated anti-mouse IgM or IgG. Controls were performed using appropriate isotype, as well as secondary antibodies alone. Cells were then subjected to flow cytometry analysis using a FACScalibur flow cytometer from Becton Dickinson (Le-Pont-de-Claix, France).

Proliferation assays

Cell growth was analyzed using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reagent (Promega, Charbonnières-les-Bains, France) according to the manufacturer's procedure. For RNA interference assay, 2 × 103 cells were seeded in 96-well plates (Thermo Fisher Scientifics, Langenselbold, Germany) after two cycles of siRNA and then cultured during 5 days in DMEM serum-free medium. Cell numbers were determined after 1, 2, 3, 4 and 5 days by adding MTS to the wells 2 h before spectrophotometric reading (absorbance at 490 nm).

For cell growth inhibition assay, transfectant cells and control cells (2.5 × 103) were seeded in 96-well plates and grown in DMEM culture medium containing 0.1% FBS. After 12 h, the medium was replaced and cells were treated for 4 days with the anti-GD2 4G2 mAb, anti-GD3 4F6 mAb or with an irrelevant mAb diluted to the indicated concentrations.

Phospho-RTK array analysis

Two days after transfection with siRNA, cells were lysed in NP-40 lysis buffer [1% NP-40, 20 mM Tris–HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate and protease inhibitor cocktail tablet (Roche, Meylan, France)]. The human Phospho-RTK array kit (R&D Systems Europe) was used according to the manufacturer's protocol. Briefly, the arrays were blocked in the appropriate blocking buffer and incubated overnight at 4°C with 200 µg of total protein extract. The arrays were washed three times and incubated with a horseradish peroxidase-conjugated phospho-tyrosine detection antibody, 1 h at room temperature, before treated with ECL-Plus Western Blotting Detection Reagent (GE Healthcare) and exposed to Kodak film (GE Healthcare).

Immunoblotting

Transfectant cells and control cells (3 × 105) were seeded in 6-well plates and grown in DMEM containing 0.1% FBS. The next day, the medium was replaced and cells were treated for 6 h with 30 µg/mL of the anti-GD2 4G2 mAb, anti-GD3 4F6 mAb or with an irrelevant mAb as a control. Cells were harvested by scraping in PBS and subjected to centrifugation (10,000 × g, 10 min). The pellets were then resuspended in lysis buffer [25 mM Tris–HCl (pH 7.4), 10 mM EDTA, 15% glycerol, 0.1% Triton X-100, protease inhibitor tablet (Roche) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich)]. The supernatants were assessed for protein concentration using the Bio-Rad RC protein assay kit II. For the phospho-Met and Met detection, 30 µg of total proteins from each cell lysate was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Molsheim, France). Membranes were then incubated overnight at 4°C with the primary antibody, incubated at room temperature for 1 h with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody. Analysis was done by chemiluminescence using the ECL-Plus western blotting detection reagent (GE Healthcare) with Kodak film.

Statistical analyses

Student's t-test was used for statistical analysis. P < 0.05 was considered as statistically significant.

Supplementary data

Supplementary data for this article is available online athttp://glycob.oxfordjournals.org/.

Funding

This work was supported by the Association pour la Recherche sur le Cancer (7936 and 5023 to P.D. and 1137 to D.T.), la Ligue régionale contre le Cancer (P.D. and D.T.) and l'ANR—Young investigator program (D.T.).

Conflict of interest

None declared.

Abbreviations

β4GalNAc T1, GM2/GD2 synthase; BSA, bovine serum albumin; c-Met, tyrosine kinase receptor for hepatocyte growth factor; DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; EGFR, epithelial growth factor tyrosine kinase receptor; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; Gal, galactose; GalNAc, N-acetylgalactosamine; GD3S, GD3 synthase; GSL, glycosphingolipid; HGF, hepatocyte growth factor; HPRT, hypoxanthine phosphoribosyltransferase; HPTLC, high-performance thin-layer chromatography; IgG, immunoglobulin G; LacCer, lactosylceramide; mAb, monoclonal antibody; MALDI, matrix-assisted laser desorption-ionization; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/ERK kinase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; Neu5Ac, N-acetylneuraminic acid; PBS, phosphate-buffered saline; PI3K, phosphoinositide-3 kinase; PR, progesterone receptor; QPCR, quantitative real-time polymerase chain reaction; RTK, tyrosine kinase receptor; SCLC, small cell lung cancer; siRNA, small interfering RNA; ST3Gal V, GM3 synthase; ST8Sia I, GD3 synthase; ST8Sia V, GT3 synthase; TOF, time-of-flight.

Acknowledgements

We thank the BioImaging Center Lille Nord-de-France (BICeL) for access to instruments and technical advice (microscopy facilities).

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Author notes

The first two authors have equally participated to this work.

Supplementary data