Overexpression of ST6GalNAcV, a ganglioside-specific α2,6-sialyltransferase, inhibits glioma growth in vivo

Abstract

Aberrant cell-surface glycosylation patterns are present on virtually all tumors and have been linked to tumor progression, metastasis, and invasivity. We have shown that expressing a normally quiescent, glycoprotein-specific α2,6-sialyltransferase (ST6Gal1) gene in gliomas inhibited invasivity in vitro and tumor formation in vivo. To identify other glycogene targets with therapeutic potential, we created a focused 45-mer oligonucleotide microarray platform representing all of the cloned human glycotranscriptome and examined the glycogene expression profiles of 10 normal human brain specimens, 10 malignant gliomas, and 7 human glioma cell lines. Among the many significant changes in glycogene expression observed, of particular interest was the observation that an additional α2,6-sialyltransferase, ST6 (α-N-acetyl-neuraminyl-2,3-β-galactosyl-1,3)-N-acetylgalactosaminide α2,6-sialyltransferase 5 (ST6GalNAcV), was expressed at very low levels in all glioma and glioma cell lines examined compared with normal brain. ST6GalNAcV catalyzes the formation of the terminal α2,6-sialic acid linkages on gangliosides. Stable transfection of ST6GalNAcV into U373MG glioma cells produced (i) no change in α2,6-linked sialic acid-containing glycoproteins, (ii) increased expression of GM2α and GM3 gangliosides and decreased expression of GM1b, Gb3, and Gb4, (iii) marked inhibition of in vitro invasivity, (iv) modified cellular adhesion to fibronectin and laminin, (v) increased adhesion-mediated protein tyrosine phosphorylation of HSPA8, and (vi) inhibition of tumor growth in vivo. These results strongly suggest that modulation of the synthesis of specific glioma cell-surface glycosphingolipids alters invasivity in a manner that may have significant therapeutic potential.

Aberrations in cell-surface glycosylation patterns are a distinguishing feature of all vertebrate tumor cells, including brain tumors (1). Cell-surface glycoproteins have been identified that are associated with the invasive potential of malignant gliomas (2). The overexpression of the glycosyltransferase, GnT-V in gliomas, led to increased invasivity of these tumor cells in vitro, suggesting that β1,6-N-acetylglucosamine–bearing N-glycans play a role in glioma invasivity (3), consistent with earlier reports showing a strong correlation between metastatic potential and the expression of tri- and tetra-antennary β1,6NAG-bearing N-glycans (4). And recently, direct demonstration of a role for the oligosaccharide component of the glioma-associated integrin, α3β1, in regulating glioma invasivity has been reported (5, 6).

Gangliosides are sialic acid-bearing glycosphingolipids that are an important part of the cell surface glycoconjugates expressed on all mammalian cells. They influence tumor growth and progression through modulation of adhesion, migration, and angiogenesis, and their expression is markedly altered in a variety of brain tumors and brain tumor model systems. Hamasaki et al. (7) have reported that GT1b ganglioside can be used as a brain metastasis marker. Mennel and Lell (8) also corroborated earlier reports that simpler ganglioside patterns were associated with the malignancy progression, but not the proliferation patterns, of astrocytomas. And a series of reports by Ladisch and coworkers (9, 10) and Nakamura et al. (11) has shown that human neuroblastomas, medulloblastomas, and astrocytomas shed gangliosides that can be detected in patient serum and cerebrospinal fluid. Li et al. (12) showed that shed ganglioside GD2 was markedly immunosuppressive and, as such, may facilitate tumor formation and progression.

We used focused 45-mer oligonucleotide microarrays coupled with quantitative RT-PCR (qRT-PCR) analyses to comprehensively query the human glyco-transcriptome (13) and found significantly lower levels of ganglioside-selective α2,6-sialyltransferase, ST6 (α-N-acetyl-neuraminyl-2,3-β-galactosyl-1,3)-N-acetylgalactosaminide α2,6-sialyltransferase 5 (ST6GalNAcV), in malignant glioblastomas (92 primary tumor specimens and 7 human glioma cell lines) relative to normal brain (34 specimens). GD1α is the terminal structure of α-series gangliosides and is synthesized from GM1b through the transfer of sialic acid to its N-acetylgalactosamine residue through an α2,6-linkage with GM1b α2,6-sialyltransferase ST6GalNAcV, which, in mice, is only active toward GM1b (12). Significant down-regulation of ST6GalNAcV has been demonstrated in lung squamous cell carcinoma (14). Its involvement in the inflammatory breast cancer phenotype (15) and in breast cancer metastases to the brain (16) has been reported. Alterations in the adhesive properties of HeLa cells by modulation of ST6GalNAcV expression has also been demonstrated (17). Interestingly, there is little to no α2,6-sialytransferase (ST6Gal1) activity, the enzyme that sialylates glycoprotein-associated, N-linked oligosaccharides, in malignant glioblastomas. Moreover, studies have shown that expression of this sialyltransferase in human glioma model systems inhibited both tumor growth and invasivity, by altering the signal transduction capability of the α3β1 integrin, because of its altered terminal sialic acid composition, and reflected in changes in focal adhesion kinase activity (5, 6).

Thus, studies were undertaken to evaluate the role of ST6GalNAcV in glioma invasivity by creating stable transfectants and evaluating them for alterations in cell-surface oligosaccharide expression, invasive potential in vitro, intracellular protein phosphorylation, and tumorigenicity in vivo.

Results

Stable Transfection.

We have demonstrated the relative absence of ST6GalNAcV mRNA in clinical gliomas and glioma cell lines, including the U373MG cell line (13). The coding region of the ST6GalNAcV gene (UnigeneID: 181819), subcloned into the pcDNA3 expression vector, was used for the generation of a panel of stable ST6GalNAcV-expressing cell lines. We chose four individually isolated clones for further study and quantitated ST6GalNAcV mRNA expression by qRT-PCR (Fig. 1). The morphology of these clones was more rounded and less dendritic than the parental U373MG cells or the control pcDNA3 transfectants. The proliferation rates of the transfectants did not significantly differ from controls, with doubling times of ≈30 h (Table S1).

Fig. 1.

ST6GalNAcV mRNA expression in U373MG/ST6GalNAcV clones. (A) Transcript abundance, normalized to GAPDH, was calculated by qRT-PCR. Data represent mean (± SD) of triplicate analyses. (**P < 0.01; ***P < 0.001; two-tailed, unpaired Student's t test). n/s, not significant (P > 0.05). (B–E) Immunohistochemical analysis after incubation with 10 μg/mL FITC-SNA (Matreya) and 1% BSA. Staining of U373MG parental cells (B), pcDNA3-transfected control cells (C), and ST6GalNAcV-transfected cells, clones S8 and S25 (D and E).

Expression of Cell Surface Glycoconjugates.

Prominent cell surface immunohistochemical staining of the transfectant clones with FITC-SNA was detectable only in the highest expressers (S8 and S25; Fig. 1). The effects of ST6GalNAcV overexpression on overall glycoprotein sialylation, evaluated by using SNA lectin Western blot analysis, demonstrated no alterations in terminal α2,6-sialylation (Fig. S1).

We compared polar lipid changes in control U373MG cells and the two highest expressing ST6GalNAc transfectants (S8 and S25) by use of a detailed, sensitive, semiquantitative method using lipid extraction, one-step nano-liquid chromatography (nLC) separation, and high-resolution mass analysis (18, 19). We observed significant modulation of specific gangliosides in the α-series pathway (Fig. 2). As ST6GalNAcV directs the synthesis of GD1α in mice, we initially expected that the increased cell surface SNA reactivity was due to specific increases in this rare, brain-specific ganglioside. The lipid profiling data clearly demonstrate that, despite no change in GD1α content in the transfectants, there was an ≈7-fold increase in another α2,6-sialoganglioside, GM2α, with a concomitant ≈3-fold decrease in GM1b and ≈2-fold decreases in Gb3 and Gb4. A 2-fold increase in GM3 was also observed in the transfectant clones. Sialic acid linked to GalNAc is thought to be relatively rare in most tissues. Although GM2α may constitute only a minor ganglioside component, the lack of appropriate standards does not allow measurement of its absolute concentration. Nonetheless, the 7-fold increase in GM2α containing the d18:1-C16:0 ceramide structure common in many tumor cells had the highest ion abundance measured across all transfectant samples (Table S2 and Fig. S2).

Fig. 2.

α-Series ganglioside profile of ST6GalNAcV transfectant (S25) via nLC-MS analysis (Left) with gangliosides demonstrating significant overexpression in the transfectants outlined in red, and those demonstrating significantly decreased expression outlined in green. The approximate fold-difference in expression is indicated. Linear quadrupole ion trap product ion mass spectrum for precursor ions of m/z 1354.783 (Right), with fragmentation pattern consistent with the structure of GM2α.

Electron-induced dissociation (EID) experiments, performed on selected ions from the nLC effluent after peak-parking to differentiate between globo-series Gb4 and iso-globoseries iGb4 isomers, confirmed the presence of Gb4 and not iGb4 (Fig. S3). EID fragmentation of Gb3/iGb3 was also performed, but because of the highly symmetrical nature of the glycan residue, we were unable differentiate between Gb3 and iGb3. Because Gb4 is biosynthesized from Gb3 and not iGb3, Gb3 is the most likely isomer detected.

In Vitro Invasivity.

The four clones examined represented a spectrum of ST6GalNAcV overexpression ranging from 2- to 25-fold. The decrease in in vitro invasivity was directly related to the level of expression of ST6GalNAc mRNA, with clones S8 and S25, the highest expressers, displaying ≈20% of the invasivity of the control cells (Fig. 3). Thus, within the limits of quantitation used in these analyses, it appears compelling that the greater the ST6GalNAcV mRNA levels, the greater the quantity of cell surface-expressed α2,6-linked glycolipids and the lesser the in vitro invasivity of the transfectants.

Fig. 3.

In vitro invasion assay. Evaluation of the relative invasivity of the transfected subclones compared with pcDNA3-transfected controls. The data are the average ± SD (bars) values of assays performed in triplicate. (**P < 0.01; ***P < 0.001; two-tailed, unpaired Student's t test). n/s, not significant (P > 0.05).

In Vitro Adhesion.

One of the primary mechanisms to modulate cellular invasivity is via differential adhesion to extracellular matrix (ECM). Compared with controls, a marked reduction in adhesion to fibronectin (≈20–40%) and increase (≈40–60%) in adhesion to laminin was observed in the two clones (S8 and S25) demonstrating comparatively high levels of ST6GalNAcV mRNA expression and cell surface α2,6-linked gangliosides (Fig. 4). No significant difference in adhesion to collagen was demonstrated.

Fig. 4.

In vitro adhesion assay. Relative adhesion of the two highest ST6GalNAcV-expressing transfectants (clones S8 and S25) on fibronectin- (A), laminin- (B), or collagen type I-coated (C) plates compared with control pcDNA3 cells. Data are average ± SEM (bars) values of three values taken from a representative experiment. n/s, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 from a Student's t test (unpaired).

Adhesion-Mediated Protein Tyrosine Phosphorylation.

Altered adhesivity of U373MG cells to fibronectin by modulation of cell surface N-linked glycoconjugates is mediated by altered tyrosine phosphorylation of p125^FAK (5). Regulation of laminin-mediated signaling in other systems has also been described (20). After adhesion to laminin, both the qualitative and quantitative pattern of phosphorylated proteins in each of the clones was identical to that observed in control cells (Fig. S4). After adhesion to fibronectin, however, there was a striking difference in the level of phosphorylation of a 75-kDa protein in all of the transfectants. The abundance of this 75-kDa phosphoprotein directly correlated with the level of ST6GalNAcV expression (Fig. 5A). The identity of this 75-kDa phosphoprotein (pp75) and the signaling pathway altered by aberrant glycolipid expression in these transfectants was examined.

Fig. 5.

Adhesion-mediated protein tyrosine phosphorylation. (A) Cells were incubated in fibronectin-coated plates, unattached cells were removed, and phosphotyrosine-modified proteins were detected as described in Methods. Lane 1, parental U373MG cells; lane 2, pcDNA3 control cells; lane 3, clone S8; lane 4, clone S23; lane 5, clone S25; lane 6, clone S32. (B) Identification of HSPA8 by nanoLC-MS/MS. The precursor ion shown is m/z 740.88, and the resultant peaks searched against the IPI human database by using both the Mascot 2.2 search engine and the SEQUEST algorithm. Eleven unique tryptic peptides, as shown, matched the heat shock cognate 71-kDa protein. (C) Immunoprecipitation analysis of phospho-HSPA8 in the ST6GalNAcV-transfected cells. Lanes 1 and 2: pcDNA3-transfected control cells and ST6GalNAcV-transfected clone (S25) were incubated in fibronectin-coated plates. HSPA8 protein content was detected by Western blot analysis using HRP-conjugated anti-HSPA8 antibody (Millipore) and visualization by ECL. Lanes 3 and 4: After adhesion to fibronectin, pcDNA3-transfected control cells and ST6GalNAcV-transfected clone (S25) were lysed in RIPA buffer, 200 μg of precleared lysate protein immunoprecipitated with antibody to HSPA8 (1B5; AbCam) and analyzed by using an antiphosphotyrosine antibody, as described in Methods.

Identification of pp75.

After Off-Gel Electrophoretic (OGE) and antiphosphotyrosine Western blot analyses, pp75 was identified in the fraction corresponding to an approximate pI range of analysis 5.3–5.6. After excision of the corresponding region of a sister Coomassie-stained gel, trypsin digestion and nano LC-MS/MS analyses identified two major proteins: the clathrin uncoating ATPase HSPA8/HSC70 (with 11 unique peptides representing 212 of 646 amino acids spanning >99.5% of the protein; Fig. 5B) and KEAP1 (with 3 unique peptides representing 66 of 624 amino acids spanning 48% of the protein). Immunoprecipitation of extracts with the respective primary antibodies followed by antiphosphotyrosine Western blot analysis demonstrated a significant increase in HSPA8 phosphorylation after adhesion to fibronectin (Fig. 5C). No change in Keap1 phosphorylation was observed (Fig. S5).

Intracranial Tumorigenicity.

Intracranial tumor growth by ST6GalNAcV-transfected U373MG (clones S8 and S25), parental U373MG, and pcDNA3 vector-transfected control cells was examined after stereotactic implantation into SCID mice (Fig. 6 A–C). Both parental and vector-transfected U373MG cells formed large tumors in vivo. Tumors formed by the ST6GalNAcV-transfected U373MG clones were, on average, 20–25% the size of those formed by vector-transfected U373MG cells (Fig. 6D).

Fig. 6.

Intracranial tumorigenicity. Glioma cells (1.25 × 10⁶) were stereotactically injected into the basal ganglia of SCID mice. Eight weeks later, brains were harvested, and sections stained with H&E (A–C). The pcDNA3 vector-transfected U373MG cells (A) formed large tumors (arrow). The ST6GalNAcV transfectants [clone S8 (B) and clone S25 (C)] formed significantly smaller tumors, with tumors primarily surrounding the needle-tract (arrows). (D) Differences in tumor maximal cross-sectional area between groups were determined by ANOVA followed by Fisher's PLSD post hoc test. Ten mice per cell line were used in each group. Data are expressed as average ± SEM (bars). (*** P < 0.0001).

Discussion

In this study, we have demonstrated that ST6GalNAcV overexpression in tumorigenic, human U373MG glioma cells leads to (i) increased expression of GM2α and GM3 gangliosides, and decreased expression of GM1b, Gb3, and Gb4, (ii) no change in α2,6-linked sialic acid-containing glycoproteins, (iii) marked inhibition of in vitro invasivity with no effect on cellular proliferation, (iv) modified cellular adhesion to fibronectin leading to significantly increased adhesion-mediated protein tyrosine phosphorylation of HSPA8 that directly correlated with decreased in vitro invasivity, and (vi) significant inhibition of in vivo tumor growth. These results strongly suggest that modulation of the synthesis of specific glioma cell-surface glycosphingolipids alters adhesion and invasivity in a manner that may play a significant role in gliomagenesis.

In mice, ST6GalNAcV encodes an enzyme catalyzing the formation of GD1α, a relatively minor brain-specific ganglioside in the “0” biosynthetic pathway (21). The lack of GD1α and the elevated expression of GM2α in the U373MG stable transfectants described here is unexpected and may result from compensatory mechanisms after long-term ST6GalNAcV expression. In humans, the “a” and “b” biosynthetic pathways are far more active than the “0” pathway, which would result in synthesis of GT1a from GD1a and GQ1b from GT1b. Because we did not detect any of these products in our lipidomic analysis, it may be more likely that the synthesis of GM2α results from altered substrate availability. Recent studies in GM3-deficient fibroblasts have demonstrated compensatory shunting of ganglioside biosynthesis toward the normally quiescent “0” pathway in murine cells but not in human cells, with differing alternative glycosphingolipid pathways used to functionally compensate for aberrant ganglioside biosynthesis (21, 22). The reduced expression of Gb3 and Gb4 observed in the transfectants is also consistent with such potential shunting of ganglioside biosynthetic pathways toward the synthesis of GM2α. At any rate, the stable overexpression of ST6GalNAcV leads to the production of nascent α2,6-linked sialoglycoconjugates on the cell surface. It also follows that the relatively low, yet significant, cell surface SNA reactivity observed in the clones exhibiting the highest levels of ST6GalNAcV expression is likely due to the low affinity of this lectin for sialic acid that is α2,6-linked to N-acetylgalactosamine, as opposed to galactose.

The modest increase in GM3 synthesis, a ganglioside possessing modulatory effects on glioma growth, adhesion, and invasivity (23), is also intriguing. At high concentrations, GM3 inhibits glioma cell proliferation predominantly by inhibition of tyrosine phosphorylation of EGF, PDGF, or FGF receptors. In our studies, no changes in cellular proliferation or in tyrosine phosphorylation of these receptors in the transfectant clones were observed. High concentrations of GM3 also inhibit tumor cell invasion predominantly via interference with integrin receptor function, which would also be reflected in events downstream of the receptor. The predominant integrin in U373MG cells is α3β1, whose primary downstream effect is tyrosine phosphorylation of p125^FAK (5). We also did not observe adhesion-mediated alterations in p125^FAK expression in the transfectants. We interpret these observations to suggest that the 2-fold increase in GM3 content in the transfectants was not sufficient to explain the differences in in vitro invasivity or inhibition of in vivo tumor growth that we have demonstrated.

In addition to their well studied roles as epitopes/cell surface markers, gangliosides participate in multiple biological functions, including inter- and intracellular signaling functions (24). Aberrantly expressed cell surface gangliosides have been demonstrated to directly impact intracellular signaling. Alteration of glycosylation in glycolipids affects intracellular localization of integrin, src, and caveolin into or out of glycolipid-enriched microdomains (25). The association of GD2 with the integrin/FAK macromolecular complex has also been demonstrated (26). GD3-mediated inhibition of laminin 5-dependent cell motility is due to disruption of specific interactions between CD9 and α3 integrin in GD3-containing microdomains (27). Ganglioside alterations in epithelial cells leading to changes in (i) adhesion to specific extracellular matrix components, (ii) relative rates of cellular proliferation and apoptosis, (iii) protease activation and function, and (iv) disruption of cell surface integrin:growth factor receptor associations have also been described (28). Significantly, GT1b-induced apoptosis in SCC12 cells by direct binding to α5β1 leads to decreased activity of the integrin-linked kinase/protein kinase B/AKT pathway (29). Although the detailed molecular mechanisms of specific glycosphingolipid modulation on glioma invasivity may differ, it is nonetheless clear that aberrant signal transduction plays a pivotal role.

The opposing effects of ST6GalNAcV overexpression on adhesion to laminin vs. fibronectin are seemingly paradoxical. Up-regulated expression of ST6GalNAcV may result in signal transduction cascades that culminate in different membrane protein expression profiles that, on one hand, are favorable to adhesion to laminin, and on the other hand, not favorable to adhesion to fibronectin. In support of this hypothesis, we did not measure any significant changes in signal transduction (measured at the level of laminin-mediated tyrosine phosphorylation) in the transfectants. Another possibility is that the effects on the receptors responsible for adhesion to different substrates may be differentially susceptible to glycolipid-associated alterations in membrane microdomains in the transfectants. Altered glioma cell adhesion to laminin has been demonstrated not to be sufficient to affect invasion (30).

Unlike the effects of adhesion to fibronectin on protein tyrosine phosphorylation in glioma cells expressing the glycoprotein ST6Gal1 (5), no decrease in p125^FAK phosphorylation was observed in these studies. There was, however, a direct relationship between the level of overexpression of ST6GalNAcV and the level of protein tyrosine phosphorylation of a unique 75-kDa (p75) protein after adhesion to fibronectin. Detailed phosphoproteomic analysis demonstrated that overexpression of ST6GalNAcV leads to enhanced de novo phosphorylation of HSPA8/HSC70 in response to attachment to fibronectin. We hypothesize that this phosphorylation is caused by the expression of GM2α ganglioside synthesized by this enzyme and plays a key role in regulating altered adhesivity. It seems reasonable to generalize that these data imply that ganglioside signal transduction is an important part of the mechanisms that underlie glioma invasivity.

Further supporting this hypothesis, it has been reported that HSPA8 is colocalized with cytoskeletal-associated proteins in the extended pseudopodial protrusions of highly invasive variants of canine kidney tumor cells (31). There is increasing evidence that phosphorylation of cell-surface-expressed heat shock proteins (HSPs) are intimately involved in tumor cell adhesion, motility, and invasion (32, 33). The effect of specific tyrosine phosphorylation of HSPA8 in mitotic and tumor cells (34) on cellular localization and function (35) has been demonstrated. In human D54MG glioma cells, siRNA-mediated knockdown of HSPA8 expression is associated with decreased glioma cell migration through vitronectin-coated membranes (36). Alteration in HSPA8 phosphorylation has been demonstrated in integrin-mediated platelet adhesion to collagen (37). The authors proposed that heat shock proteins, including HSPA8, act as signaling scaffolds regulating platelet adhesion and spreading (38). HSPA8 is coexpressed with, and anchored by, cytoskeletal β-actin on the cell surface of HTLV-susceptible T cells and is an integral part of a complex supported by palmitoyl (16:0)-oleoyl (18:1)-phosphatidylglycerol in the lipid bilayer (39). The N-terminal ATPase-containing domain of HSPA8 involved in uncoating of clathrin-coated vesicles has been shown to directly bind to cell surface 3′-sulfogalactolipids, sulfogalactosyl ceramide, and sulfogalactoglycerolipid (40). Because membrane localization of HSPs appears to be restricted to lipid rafts/glycosynapses on transformed cells (41) and is specifically associated with Gb3 in colon and pancreatic tumor cells (42), perhaps the differential phospho-HSPA8-mediated signal transduction modulated by specific membrane ganglioside content provides the scaffold for signal transduction cascades in glial tumors.

Hakomori (43, 44) has discussed models of how altered membrane glycosphingolipids (GSL) can influence the tumorigenic phenotype, based on their ability to form clusters at the cell surface and interact with various functional components on the cell membrane. These clusters (or “glycosynapses”) interact with such functional membrane proteins, including integrins, growth factor receptors, tetraspanins, and nonreceptor cytoplasmic protein kinases (e.g., src kinases and small G proteins) that ultimately control GSL-modulated cell adhesion, growth, and motility. It is through this framework that modulation of cell surface carbohydrate expression and subsequent disorganization of cell membrane components can mediate cell signaling, leading to changes in cellular phenotype.

From the “glycosynapse” perspective, our results can be summarized in the following way: Manipulation of the synthesis of cell surface gangliosides by ST6GalNAcV gene transfer alters the composition of specific membrane domains. Alteration in the composition of specific membrane domains, in turn, alters adhesivity to ECM components, increases the phosphorylation of HSPA8 protein, and decreases the invasive potential and in vivo tumorigenicity of glioma cells. Based on this and previous studies (3, 5, 6), modulating the expression of α2,6-sialic acids on either gangliosides or the N-linked oligosaccharides expressed on cell surface signaling molecules (i.e., integrins) by modulating glyco-gene expression may have therapeutic potential for the treatment of malignant gliomas.

Methods

Cell Culture and Stable Transfection.

Cells were maintained exactly as described (5). The 1.3-kb protein coding region of the human ST6GalNAcV gene (UnigeneID: 181819), subcloned into the pcDNA3 expression vector, was transfected into human U373MG glioma cells, and G418-resistant transfectants screened for ST6GalNAcV expression by qRT-PCR.

Immunohistochemistry.

Cell surface α2,6-linked sialoglycoconjugate expression was confirmed by using fluorescein isothiocyanate (FITC)-conjugated Sambucus nigra agglutinin (SNA) (Matreya) exactly as described (5). SNA recognizes epitopes containing α2,6-linked sialic acid linked to either Gal or GalNAc (45).

SNA Lectin Blot Analysis.

Expression of cell-surface glycoproteins was evaluated by SNA lectin blot analysis, as described (5), by using the DIG Glycan Differentiation Kit (Roche Molecular Biochemical), exactly as per the manufacturer's recommendations.

nLC-MS Detection of Polar Lipids.

Polar lipids were extracted from cells and analyzed by nLC-MS as described (18, 19) and detailed in SI Methods. Data-dependent MS/MS was performed in the linear quadrupole ion trap (CID) during collection of the ICR time-domain data. Mean fold change was calculated by direct comparison of average ion signal magnitude in transfectants vs. control cells, the combined data representing different ceramide chain compositions. To differentiate between Gb4/iGb4 isomers, EID experiments were also performed as detailed in SI Methods.

Invasion Assay.

In vitro invasivity was examined by using Biocoat Matrigel Invasion Chambers, exactly as described (5).

Cell Adhesion Assay.

Cell adhesion to human fibronectin, laminin, and collagen type I was measured, also as described (5).

Adhesion-Mediated Protein Tyrosine Phosphorylation.

Cells were incubated on fibronectin-coated plates, unattached cells were removed, and Western blot analysis was performed by using a HRP-conjugated antiphosphotyrosine antibody (clone 4G10; Millipore) performed as described (5).

Phosphoproteomic Analysis.

After adhesion to fibronectin as above, 300 μg of cell lysate protein was fractionated on an Agilent 3100 OFFGel Electrophoresis Apparatus by using 24-cm IPG strips, pI 3–10 (GE Healthcare) in urea, thiourea, DTT, glycerol, and the corresponding GE ampholine under conditions recommended by Agilent. The buffer in each well, containing focused proteins, was recovered for Western blot analyses to identify appropriate phosphoprotein-containing fractions by using an antiphosphotyrosine antibody. Protein identification in the corresponding band, excised from Coomassie-stained sister gels, was performed by nanoLC-MS/MS of tryptic fragments, as described (46) on a Thermo Instruments LTQ-FT equipped with a Dionex Ultimate 3000 2D microcapillary HPLC system.

Immunoprecipitation.

After adhesion to fibronectin-coated plates, cells were lysed in RIPA buffer and 200 μg of precleared lysate protein was incubated with 5 μg/mL antibody to HSPA8 (1B5; AbCam). After incubation with Protein A/G Plus Agarose (Santa Cruz Biotechnology), beads were washed, denatured, and analyzed by Western blot analysis using antiphosphotyrosine antibody, as above.

Intracranial Tumorigenicity.

Intracranial tumorigenicity of ST6GalNAcV-, pcDNA3 vector-transfected cells, and parental U373 MG cells was examined in SCID mice (C.B-17 scid/scid, 6 wk of age; Charles River Laboratories), as described (6) and detailed in SI Methods. All mice were maintained in the animal facility of Northwestern University, Evanston, IL, and approved by their Institutional Animal Care and Use Committee.