Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide-or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e. buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding.

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Two membrane subfractions, one enriched in GM3 ganglioside and the other containing caveolin, were separated from low density detergent-insoluble membrane fraction prepared by sucrose density gradient centrifugation of postnuclear fraction of mouse melanoma B16 cells. The GM3-enriched subfraction, separated by anti-GM3 monoclonal antibody DH2, contained sphingomyelin, cholesterol, c-Src, and Rho A but not caveolin. In contrast, the caveolin-containing subfraction, separated by anti-caveolin antibody, contained neither GM3, c-Src, nor Rho A but did contain glucosylceramide, Ras, a very small quantity of sphingomyelin, and a very large quantity of cholesterol. The GM3/c-Src-enriched membrane subfraction was characterized by (i) maintenance of GM3-dependent adhesion and (ii) susceptibility to being activated for signal transduction through GM3.32 P-Phosphorylation of c-Src (M r 60,000) together with two other components (M r 45,000 and 29,000) was enhanced in the fraction bound to dishes coated with asialo-GM2 (Gg3) or with anti-GM3 monoclonal antibody DH2, detected by incubation with [␥-32 P]ATP at 37°C for 5 min. GM3-dependent adhesion of B16 cells to Gg3-coated dishes and associated signaling were not reduced or abolished in the presence of either filipin or nystatin, which are cholesterol-binding reagents known to abolish caveolae structure and function. B16 melanoma cells incubated with filipin (0.16 -0.3 g/ml) or with nystatin (25 g/ml) for 30 min showed depletion of cholesterol in detergentinsoluble membrane fraction but were still capable of binding to Gg3-coated plate and capable of the associated signaling. Thus, the GM3-enriched subfraction, involved in cell adhesion and capable of sending signals through GM3, represents a membrane domain distinguishable from caveolin-containing subfraction or caveolae. This microdomain is hereby termed the "glycosphingolipid signaling domain" or "glycosignaling domain".

Epidermal growth factor receptor (EGFR) at membrane microdomains plays an essential role in the growth control of epidermal cells, including cancer cells derived therefrom. Ligand-dependent activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, but to a much lesser degree by other glycosphingolipids. However, the mechanism of the inhibitory effect of GM3 on EGFR tyrosine kinase has been ambiguous. The mechanism is now defined by binding of N-linked glycan having multiple GlcNAc termini to GM3 through carbohydrate-to-carbohydrate interaction, based on the following data: (i) EGFR (molecular mass, Ϸ170 kDa) has N-linked glycan with GlcNAc termini, as probed by mAb (J1) or lectin (GS-II); (ii) GS-II-bound EGFR also bound to anti-EGFR Ab as well as to GM3-coated beads; (iii) GM3 inhibitory effect on EGFR tyrosine kinase was abrogated in vitro by coincubation with glycan having multiple GlcNAc termini and in cell culture in situ incubated with the same glycan; and (iv) cells treated with swainsonine, which increased expression of complex-type and hybridtype glycans with GlcNAc termini, displayed higher inhibition of EGFR kinase by GM3 than swainsonine-untreated control cells. A similar effect was observed with 1-deoxymannojirimycin, which increased hybrid-type structure in addition to major accumulation of high mannose-type glycan. These findings indicate that N-linked glycan with GlcNAc termini linked to EGFR is the target to interact with GM3, causing inhibition of EGF-induced EGFR tyrosine kinase.carbohydrate-to-carbohydrate interaction ͉ N-linked glycan ͉ glycosphingolipid ͉ ganglioside ͉ oligosaccharide Fr.B

Previous studies demonstrated that certain glycosphingolipids (GSLs) are involved in various cell functions, such as cell growth and motility. Recent studies showed changes in GSL expression during differentiation of human embryonic stem cells; however, little is known about expression profiles of GSLs in cancer stem cells (CSCs). CSCs are a small subpopulation in cancer and are proposed as cancer-initiating cells, have been shown to be resistant to numerous chemotherapies, and may cause cancer recurrence. Here, we analyzed GSLs expressed in human breast CSCs by applying a CSC model induced through epithelial-mesenchymal transition, using mass spectrometry, TLC immunostaining, and cell staining. We found that (i) Fuc-(n)Lc4Cer and Gb3Cer were drastically reduced in CSCs, whereas GD2, GD3, GM2, and GD1a were greatly increased in CSCs; (ii) among various glycosyltransferases tested, mRNA levels for ST3GAL5, B4GALNT1, ST8SIA1, and ST3GAL2 were increased in CSCs, which could explain the increased expression of GD3, GD2, GM2, and GD1a in CSCs; (iii) the majority of GD2+ cells and GD3+ cells were detected in the CD44 hi /CD24 lo cell population; and (iv) knockdown of ST8SIA1 and B4GALNT1 significantly reduced the expression of GD2 and GD3 and caused a phenotype change from CSC to a non-CSC, which was detected by reduced mammosphere formation and cell motility. Our results provide insight into GSL profiles in human breast CSCs, indicate a functional role of GD2 and GD3 in CSCs, and suggest a possible novel approach in targeting human breast CSCs to interfere with cancer recurrence.

Epithelial-to-mesenchymal cell transition (EMT) is a basic process in embryonic development and cancer progression. The present study demonstrates involvement of glycosphingolipids (GSLs) in the EMT process by using normal murine mammary gland NMuMG, human normal bladder HCV29, and human mammary carcinoma MCF7 cells. Treatment of these cells with D-threo-1-(3 ,4 -ethylenedioxy)-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), the glucosylceramide (GlcCer) synthase inhibitor, which depletes all GSLs derived from GlcCer, (i) down-regulated expression of a major epithelial cell marker, E-cadherin; (ii) up-regulated expression of mesenchymal cell markers vimentin, fibronectin, and N-cadherin; (iii) enhanced haptotactic cell motility; and (iv) converted epithelial to fibroblastic morphology. These changes also were induced in these cell lines with TGF-␤, which is a well-documented EMT inducer. A close association between specific GSL changes and EMT processes induced by EtDO-P4 or TGF-␤ is indicated by the following findings: (i) The enhanced cell motility of EtDO-P4-treated cells was abrogated by exogenous addition of GM2 or Gg4, but not GM1 or GM3, in all 3 cell lines. (ii) TGF-␤ treatment caused changes in the GSL composition of cells. Notably, Gg4 or GM2 was depleted or reduced in NMuMG, and GM2 was reduced in HCV29. (iii) Exogenous addition of Gg4 inhibited TGF-␤-induced changes of morphology, motility, and levels of epithelial and mesenchymal markers. These observations indicate that specific GSLs play key roles in defining phenotypes associated with EMT and its reverse process (i.e., mesenchymal-to-epithelial transition).E-cadherin ͉ EtDO-P4 ͉ Gg4 ͉ motility ͉ TGF-beta E pithelial cells in tissues or cultured in vitro change their morphology, growth behavior, and motility when they encounter a different microenvironment. For example, epithelial cells that come in contact with soluble extracellular matrix components acquire characteristics similar to those of mesenchymal fibroblasts, as originally observed by Hay and colleagues (1-3). After extensive further studies, a concept emerged that the transitional process from epithelial to mesenchymal phenotype is commonly associated with embryonic development, as well as pathological conditions, such as fibrosis or cancer metastasis, and the process was termed ''epithelial-to-mesenchymal transition'' (EMT) (4-10).The EMT process has been characterized, in addition to cell morphology change and increased cell motility, by a striking decline in epithelial markers, such as E-cadherin, desmoplakin, and cytokeratins, accompanied by enhanced expression of mesenchymal markers, such as vimentin, fibronectin, and N-cadherin (10, 11).Previous studies from our group and others have demonstrated that glycosphingolipids (GSLs) mediate cell adhesion (12-15), and they modulate cell growth through their effect on growth factor receptor tyrosine kinases (16)(17)(18)(19)(20). In addition, we have demonstrated that some GSLs, particularly gangliosides, play an essential role...

Glycosphingolipids (GSLs) at the cell surface membrane are associated or complexed with signal transducers (Src family kinases and small G-proteins), tetraspanins, growth factor receptors, and integrins. Such organizational framework, defining GSL-modulated or -dependent cell adhesion, motility, and growth, is termed "glycosynapse" (Hakomori, S., and Handa, K.

The process termed "epithelial-mesenchymal transition" (EMT) was originally discovered in ontogenic development, and has been shown to be one of the key steps in tumor cell progression and metastasis. Recently, we showed that the expression of some glycosphingolipids (GSLs) is down-regulated during EMT in human and mouse cell lines. Here, we demonstrate the involvement of GalNAc-type (or mucintype) O-glycosylation in EMT process, induced with transforming growth factor β (TGF-β) in human prostate epithelial cell lines. We found that: (i) TGF-β treatment caused up-regulation of oncofetal fibronectin (onfFN), which is defined by mAb FDC6, and expressed in cancer or fetal cells/tissues, but not in normal adult cells/tissues. The reactivity of mAb FDC6 requires the addition of an O-glycan at a specific threonine, inside the type III homology connective segment (IIICS) domain of FN. (ii) This change is associated with typical EMT characteristics; i.e., change from epithelial to fibroblastic morphology, enhanced cell motility, decreased expression of a typical epithelial cell marker, E-cadherin, and enhanced expression of mesenchymal markers. (iii) TGF-β treatment up-regulated mRNA level of FN containing the IIICS domain and GalNAc-T activity for the IIICS domain peptide substrate containing the FDC6 onfFN epitope. (iv) Knockdown of GalNAc-T6 and T3 inhibited TGF-β-induced up-regulation of onfFN and EMT process. (v) Involvement of GSLs was not detectable with the EMT process in these cell lines. These findings indicate the important functional role of expression of onfFN, defined by sitespecific O-glycosylation at IIICS domain, in the EMT process.O-glycosylated fibronectin | siRNA G lycoconjugates, such as glycosphingolipids (GSLs) and N-and O-linked glycoproteins, have been shown to play important roles in embryogenesis (1), tumorigenesis, and cancer progression (2). Specific types of glycosyl residues modulate particular signaling pathways and regulate cell phenotypes. For example, GM3 inhibits epithelial growth factor receptor (EGFR) activation (3, 4); GM2 associated with tetraspanine CD82 inhibits the activation of the hepatocyte growth factor receptor cMet (5); and O-Fucose glycans modulate Notch signaling, which controls the fate of many cell types (6-8).Epithelial-mesenchymal transition (EMT) was initially observed during early embryonic development and organ formation (9-11). Accumulating evidence has shown that the EMT process plays a key role in disease development, particularly in cancer progression to metastasis (10-14) and fibrosis (15). During EMT, cells lose their apical-basal polarity, change morphology to fibroblastic, display reduced expression of epithelial cell marker molecules such as E-cadherin (Ecad), and enhance expression of mesenchymal cell marker molecules such as fibronectin (FN), N-cadherin (Ncad), vimentin, and matrix-metalloproteinases (MMPs). When combined, these effects result in increased cell motility (10-15).Our recent studies found a reduction in specific GSLs (Gg4 and/ or GM2) in ...