Molecules in the signaling pathway activated by gangliosides can be targets of therapeutics for malignant melanomas

There have been a number of studies on tumor-specific glycolipid antigens. In particular, neuroectoderm-derived cancers express characteristic ganglioside antigens, some of them have been used as tumor markers and/or target of immunotherapy. Molecules in the signaling pathway activated by gangliosides have been analyzed. Here, we reported results on the functions of molecules involved in the signaling pathway to enhance malignant properties of human melanomas under GD3 expression, and emphasized that those molecules including tumor-associated antigens can be targets of therapeutics for malignant melanomas.     

Glycosphingolipid-dependent cross-talk between glycosynapses interfacing tumor cells with their host cells: essential basis to define tumor malignancy

Status of tumor progression (either remaining in situ, or becoming invasive/metastatic) may be defined largely by subtle interactions ('cross-talk') in a microenvironment formed by interfacing tumor cell and host cell membrane domains (termed 'glycosynapses') involved in glycosylation-dependent cell adhesion and signaling. Functional roles of tumor-associated gangliosides, organized in glycosynapses of three types of tumor cell lines, are discussed. Gangliosides function as adhesion receptors or as 'sensors' that can be stimulated by antibodies, with consequent activation of signal transducers leading to enhanced motility and invasiveness.     

TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.     

Roles of plasma membrane-associated sialidase NEU3 in human cancers

Altered sialylation of glycosphingolipids is observed in cancer as a ubiquitous phenotype, leading to the appearance of tumor-associated antigens, aberrant adhesion and disturbance of transmembrane signaling. To understand the pathological significance of aberrant alterations of gangliosides in cancer, our studies have been focused on sialidase, which is responsible for the removal of sialic acids from glycoproteins and glycolipids. Among human sialidases so far identified, sialidase NEU3 is a key enzyme for ganglioside degradation because of its uniqueness both in its localization in the plasma membrane and in specifically hydrolyzing gangliosides. NEU3 is markedly up-regulated in many types of cancers including colon and renal carcinomas and suppresses apoptosis of cancer cells. The present paper briefly summarizes our recent results on the sialidase alterations and their significance in cancer. NEU3 is indeed closely related to malignancy and thus may be a potential target for cancer diagnosis and therapy.     

Induction of GM1a/GD1b synthase triggers complex ganglioside expression and alters neuroblastoma cell behavior; a new tumor cell model of ganglioside function

Neuroblastoma is the most common extracranial solid tumor in children and tumor ganglioside composition has been linked to its biological and clinical behavior. We recently found that high expression of complex gangliosides that are products of the enzyme GM1a/GD1b synthase predicts a more favorable outcome in human neuroblastoma, and others have shown that complex gangliosides such as GD1a inhibit metastasis of murine tumors. To determine how a switch from structurally simple to structurally complex ganglioside expression affects neuroblastoma cell behavior, we engineered IMR32 human neuroblastoma cells, which contain almost exclusively (89%) the simple gangliosides (SG) GM2, GD2, GM3, and GD3, to overexpress the complex gangliosides (CG) GM1, GD1a, GD1b and GT1b, by stable retroviral-mediated transduction of the cDNA encoding GM1a/GD1b synthase. This strikingly altered cellular ganglioside composition without affecting total ganglioside content: There was a 23-fold increase in the ratio of complex to simple gangliosides in GM1a/GD1b synthase-transduced cells (IMR32-CG) vs. wild type (IMR32) or vector-transfected (IMR32-V) cells with essentially no expression of the clinical neuroblastoma marker, GD2, confirming effectiveness of this molecular switch from simple to complex ganglioside synthesis. Probing for consequences of the switch, we found that among functional properties of IMR32-CG cells, cell migration was inhibited and Rho/Rac1 activities were altered, while proliferation kinetics and cell differentiation were unaffected. These findings further implicate cellular ganglioside composition in determining cell migration characteristics of tumor cells. This IMR32 model system should be useful in delineating the impact of ganglioside composition on tumor cell function.     

Ganglioside GD1a enhances VEGF-induced endothelial cell proliferation and migration

Tumor progression requires normally quiescent endothelial cells to form new vascular networks. This angiogenesis is dependent upon several soluble factors, prominent among which is vascular endothelial growth factor (VEGF). Other tumor-associated molecules, such as gangliosides, sialic acid-containing glycosphingolipids expressed by tumor cells and shed into the tumor microenvironment, may also modulate tumor angiogenesis. Here we assessed the influence of a highly purified ganglioside, G(D1a), on responses of normal human umbilical vein endothelial cells (HUVEC) to VEGF. Preincubation of HUVEC with G(D1a) enhanced VEGF-induced cell proliferation; 10 microM G(D1a) caused a twofold increase in DNA synthesis. The migration of HUVEC across a VEGF gradient was also enhanced by 50%, even with only a brief (1 h) preexposure of the cells to the same concentration of G(D1a). These findings suggest that gangliosides shed by tumor cells can promote tumor angiogenesis by enhancing the VEGF response of endothelial cells in the tumor microenvironment.     

Immunology of gangliosides

This review discusses the immunology of gangliosides from the perspective of tumor, neuronal and general immunology. Antiganglioside antibodies in human sera are invariably IgM and are found in healthy individuals. Their titers decline with age. Persistent high titer of IgM is associated with several diseases, particularly neuropathies. Membrane-bound gangliosides are important tumor-associated antigens and targets for immune attack. Cells enriched with gangliosides can be used as cancer vaccines. Efficacy of these vaccines depends on the viability of whole cells, integrity of the cell membranes, adjuvants and topography of the tumor-associated antigens. The role of antiganglioside IgM is to eliminate the immunosuppressive gangliosides shed from tissues during ageing, degeneration of neural and extraneural tissues, and tumor growth and necrosis. In addition, in vitro observations with human and murine monoclonal antibodies suggest that they are capable of complement dependent cytotoxicity and apoptosis.     

Tumor gangliosides inhibit the tumor-specific immune response.

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5-20 microM inhibited the tumor-specific secondary proliferative response (80-100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63-74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.     

Alteration of ganglioside composition by stable transfection with antisense vectors against GD3-synthase gene expression.

Gangliosides are ubiquitous components of mammalian cells. Their expression is frequently altered in many tumor types. We previously showed that alteration of the ganglioside composition often resulted in changes in cellular morphology and differentiation of cultured cells. In this study, we targeted sialyltransferase gene expression by the antisense knockdown experiment, and the results showed that inhibition of the expression of gangliosides GD3 and O-acetylated GD3 (OAc-GD3) in the neuroblastoma F-11 cells greatly reduced the tumor growth in nude mice. The sense and antisense vectors containing either a 5' end fragment or the entire sequence of the cDNA coding for GD3-synthase were prepared and used in separate experiments to transfect the F-11 cells which express high levels of gangliosides GD3 and OAc-GD3. Single clones were isolated and expanded. Both the activity of the GD3-synthase and the concentrations of GD3 and OAc-GD3 in the antisense-transfected cells were dramatically decreased as a result of transfection with the antisense expression vectors. Further characterization of the antisense-transfected cells showed reduced rates of cell growth and neurite formation and changes in cellular morphology. When the cells were inoculated in athymic nude mice, the tumor growth rate was remarkably suppressed although the tumor incidence was not affected by the altered ganglioside composition. These results indicate that the tumor-associated ganglioside(s) is(are) involved in regulation of tumor growth, probably through the stimulation of angiogenesis of the tumor.     

Cellular gangliosides promote growth factor-induced proliferation of fibroblasts

Cell surface gangliosides have been proposed as modulators of transmembrane signaling. In this study, we used two complementary approaches to investigate the function of cellular gangliosides in the response of mammalian fibroblasts to growth factors. First, inhibition of glucosyl ceramide synthase by a new specific inhibitor of d-l-threo-1-phenyl-2-hexadecanoylamino-3 -pyrrolidino-1-propanol-HC l (glucosylceramide synthase), which depletes cellular gangliosides at a concentration of 1 microm without causing an increase in ceramide levels, blocked epidermal growth factor-stimulated proliferation of fibroblasts. Similarly, responses to several other growth factors that activate receptor tyrosine kinases, including fibroblast growth factor, insulin-like growth factor-I, and platelet-derived growth factor, were inhibited by 50-100%. Conversely, enrichment of cellular gangliosides by preincubation of the mouse and human fibroblasts with exogenously added gangliosides enhanced growth factor-elicited cell proliferation. Novel findings of this study, distinguishing it from previous similar studies, include differential effects of preincubation versus continuous incubation of cells with gangliosides on growth factor-dependent cell proliferation and the growth factor-like action of NeuNAc alpha 2-3Gal beta 1-3GalNAc beta 1-4(NeuNAc alpha 2-3)Gal beta 1-4Glc beta 1-1Cer when cells are pretreated with the ganglioside.     

Shedding of Gangliosides by Human Medulloblastoma Cells

Shedding of immunosuppressive gangliosides is an important characteristic of both experimental and human tumors. Using a medulloblastoma cell line, Daoy, with a very high ganglioside expression (141 ± 13 nmol/10⁸cells) and a well-characterized ganglioside complement, we have now studied ganglioside shedding by human brain tumor cells. Shedding of gangliosides, quantified by metabolic radiolabeling, was significant (169 pmol/10⁸cells/h) and was generalized with respect to the major ganglioside carbohydrate structures (G_M2, G_M3, and G_D1a). For each ganglioside, however, shedding was selective for ceramide structures containing shorter fatty acyl chains. Rapid and ceramide-selective shedding was confirmed in two additional human medulloblastoma cell lines, D341 Med and D283 Med (112 and 59 pmol/10⁸cells/h). Significant ganglioside shedding is therefore a common characteristic of human medulloblastoma cells and may influence the biological behavior of this tumor, in view of immunosuppressive and other biological properties of shed gangliosides.     

Ganglioside modulation regulates epithelial cell adhesion and spreading via ganglioside-specific effects on signaling

Gangliosides are implicated in regulating cell adhesion and migration on fibronectin by binding with the alpha(5) subunit of alpha(5)beta(1) integrin. However, the effects of gangliosides on cell spreading and related signaling pathways are unknown. Increases in gangliosides GT1b and GD3 inhibited spreading on fibronectin, concurrent with inhibition of Src and focal adhesion kinase. Although antibody blockade of GT1b or GD3 function and gene-modulated ganglioside depletion stimulated spreading and activated Src and focal adhesion kinase, the augmented spreading by disruption of GT1b function, but not by disruption of GD3 function, was inhibited by blockade of Src and focal adhesion kinase activation. In contrast, inhibitors of protein kinase C prevented the stimulation of spreading by GD3 functional inhibition, but not by GT1b functional blockade. Modulation of either GT1b or GD3 content affected phosphoinositol 3-kinase activation, and inhibition of this activation reversed the stimulation of cell spreading by anti-GD3 antibody, anti-GT1b antibody, and ganglioside depletion, suggesting that phosphoinositol 3-kinase is an intermediate in both the FAK/Src and protein kinase C pathways that lead to cell spreading. These studies demonstrate that epithelial cell ganglioside GT1b modulates cell spreading through alpha(5)beta(1)/FAK and phosphoinositol 3-kinase signaling, whereas GD3-modulated spreading appears to involve phosphoinositol 3-kinase-dependent protein kinase C signaling.     

Localization of the ganglioside-binding site of fibronectin

It has been demonstrated via biological assays that fibronectin possesses a receptor for gangliosides that is involved in cell adhesion and restoration of the normal morphology of transformed cells. In this study, fluorescence polarization has been employed to monitor the binding of ganglioside oligosaccharide to fibronectin. Parameters involved in ganglioside oligosaccharide binding to fibronectin are described and compared to the interaction of heparin with fibronectin. A Kd of 1.4 X 10(-8) mol/liter has been calculated, and it is demonstrated that labeled ganglioside oligosaccharides can be eluted from fibronectin with either unlabeled ganglioside oligosaccharides or 4 M urea. Using the fluorescence polarization assay developed in this study for measurement of ganglioside binding to fibronectin, it is demonstrated that gangliosides bind to the 31,000-dalton amino terminal heparin-binding domain of fibronectin. A ganglioside-Sepharose affinity column has been constructed which specifically binds the 31,000-dalton amino terminal fragment of fibronectin. The localization of the ganglioside receptor to the amino terminal domain of fibronectin indicates that the ganglioside receptor is distinct from the putative fibronectin cell surface receptor which is located near the center of the fibronectin molecule.     

Uptake and metabolism of exogenous glycosphingolipids by cultured cells

Exogenous glycosphingolipids, especially gangliosides, are used to study transport and metabolism of their endogenous counterparts as well as their role in cell adhesion, cell recognition and signal transduction. Unlike monodispersed solutes, in aqueous media ganglioside molecules aggregate into micelles (or bilayer structures) with a very low critical micellar concentration. Upon addition to cells in culture, exogenous gangliosides bind to the cell surface in three operationally defined modes: loosely associated micelles removable by serum; tightly attached micelles removable by proteases such as trypsin; and ganglioside molecules inserted into the outer leaflet of the plasma membrane. As shown by a biotin-labeled derivative of the ganglioside GM1 these inserted molecules are endocytosed and transported to intralysosomal membranes for catabolism. The benefit from using (partially) nondegradable as well as semi-truncated glycosphingolipids in transport studies is discussed.     

III6NeuAcLc4Cer in human SW1116 colorectal carcinoma cells: a possible oncofetal antigen that is not dependent on Lewis gene expression

Monospecific rabbit antibodies directed against the human milk sialyloligosaccharides III6NeuAcLcOse4 (sialyltetrasaccharide b), IV3NeuAcLcOse4 (sialyltetrasaccharide a), and IV6NeuAcnLc4Ose (sialyltetrasaccharide c) were used to detect their homologous haptens as gangliosides or ganglioside-derived sialyloligosaccharides from the human colorectal carcinoma cell line SW1116. III6NeuAcLc4Cer was first detected in human meconium [P. A. Prieto and D. F. Smith (1985) Arch. Biochem. Biophys. 241, 281-289], and its presence in a total ganglioside fraction of SW1116 cells together with its absence from a total lipid extract of normal human intestinal mucosa are consistent with III6NeuAcLc4Cer being a tumor-associated oncofetal antigen. IV3NeuAcLc4Cer, a ganglioside in human meconium [P. A. Prieto and D. F. Smith (1986) Arch. Biochem. Biophys. 249, 243-253], was also detected in SW1116 cells; an observation that is consistent with its being the immediate precursor to the sialyl-Lea ganglioside in SW1116 cells. Specific antisera against sialylated type 1 oligosaccharide chains whose expression is independent of the Lewis gene fucosyltransferase may be useful diagnostic reagents for oncofetal, carbohydrate antigens.     

Synthesis, Shedding, and Intercellular Transfer of Human Medulloblastoma Gangliosides: Abrogation by a New Inhibitor of Glucosylceramide Synthase

Abstract: The biological activity of human medulloblastoma tumor gangliosides very likely involves the interaction of these molecules with host cells in the tumor microenvironment. To trace the hypothesized intercellular transfer of shed medulloblastoma gangliosides, we used an in vitro dual-chamber culture system in which the tumor cells, the shed gangliosides, and the target cells to which they might bind would not be perturbed during the transfer process. We observed that under these unmanipulated conditions, gangliosides were shed by the Daoy medulloblastoma cell line (∼300 pmol/10⁸ cells/h), traversed the chamber membrane, and stably bound to the target fibroblasts at the very high density of 10⁷ molecules per cell within 48 h. To determine if this substantial intercellular transfer of shed gangliosides, with its potential of modifying target cell function, could be blocked, we evaluated a new inhibitor of glucosylceramide synthase, dl - threo -1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP). PPPP (1.0 µ M ) reduced (90%) Daoy cell ganglioside content strikingly, without causing toxicity or inhibiting cell proliferation. Subsequently, ganglioside shedding by the medulloblastoma cells was diminished significantly (to ∼50 pmol/10⁸/h), and binding of radiolabeled shed medulloblastoma gangliosides to target fibroblasts was consequently almost completely abrogated. We conclude that the shedding and transfer of potentially biologically active human medulloblastoma gangliosides can be diminished effectively by PPPP.     

Disialyl gangliosides enhance tumor phenotypes with differential modalities

Sialic acid-containing glycosphingolipids, gangliosides are highly expressed in human cancer cells and regulate cell signals transduced via membrane microdomains. Generally, disialyl gangliosides enhance tumor phenotypes, while monosialyl gangliosides suppress them. In particular, gangliosides GD3 and GD2 are highly expressed in melanomas and small cell lung cancer cells, and their expression cause increased cell growth and invasion. In osteosarcomas, expression of GD3 and GD2 also enhanced cell invasion and motility, and caused increased phosphorylation of focal adhesion kinase and paxillin. In addition to focal adhesion kinase, Lyn kinase was also activated by GD3/GD2 expression, leading to the phosphorylation of paxillin. In contrast with melanoma cells, osteosarcomas showed reduced cell adhesion with increased phosphorylation of paxillin. Thus, increased expression of GD3/GD2 caused enhanced activation of signaling molecules, leading to distinct phenotypes between melanomas and osteosarcomas, i.e. increased and decreased adhesion activity. Thus, whole features of glycolipid-enriched microdomain/rafts formed in the individual cancer types seem to determine the main signaling pathway and biological outcome.     

Enhancement of epidermal growth factor signaling and activation of SRC kinase by gangliosides

In a recent study, inhibition of cellular ganglioside synthesis blocked growth factor-induced fibroblast proliferation. Conversely, enrichment of cell membrane gangliosides by ganglioside preincubation enhanced growth factor-elicited cell proliferation. In the absence of serum and growth factors, NeuNAcalpha2-3Galbeta1-3GalNAcbeta1-4(NeuNAcalpha2-3)Galbeta1-4Glcbeta1-1Cer (G(D1a)) acted like a growth factor when cells were pretreated with the ganglioside, stimulating proliferation of normal human dermal fibroblasts and Swiss 3T3 fibroblasts. In contrast, growth inhibition was observed when high concentrations of gangliosides were continuously present in the culture medium during incubation of fibroblasts with growth factors (Li, R., Manela, J., Kong, Y., and Ladisch, S. (2000) J. Biol. Chem. 275, 34213-34223). Here, we investigated the mechanisms whereby gangliosides elicit proliferation-coupled signaling in normal human dermal fibroblasts. Incubation of the fibroblasts with G(D1a) enhanced epidermal growth factor (EGF) receptor autophosphorylation and Ras and MAPK activation in a dose-dependent manner. Exposure of the cells to G(D1a) also enhanced the phosphorylation of Elk-1 by the activated MAPK. Brief pretreatment of the cells with PD98059 blocked the enhancing effect of gangliosides on EGF-induced MAPK activation. In the absence of serum and growth factors, G(D1a) incubation induced phosphorylation of Src kinase, Ras activation, and phosphorylation of MAPK and Elk-1 in a dose-dependent manner. The activation of Src kinase was confirmed by enhanced Src kinase activity. Brief treatment of the cells with PP1 blocked the activation of Src kinase and MAPK. Again, PD98059 treatment inhibited ganglioside-elicited MAPK phosphorylation. Among the gangliosides tested, G(D1a), was the most active molecule, whereas lactosylceramide was the least active one, indicating relative structural specificity of the ganglioside action. In conclusion, gangliosides promote fibroblast proliferation through enhancement of growth factor signaling and activation of Src kinase.     

Localization and functional role of gangliosides from nerve tissue of vertebrates

Gangliosides in the animal organism are typical components of plasma membranes of nerve cells in which the concentration of polysialogangliosides is especially high. The high concentration of tri- and tetrasialoganyliosides and ganglioside GD1b is peculiar to primary cultures of nerve cells, whereas these gangliosides are practically not present in the culture of the transformed nerve cells which have lost their ability to sinaptogenesis, they are not found in cultures of oligodendro- and astroglia as well. The addition of exogenic gangliosides to nerve cells cultures stimulates the formation of processes in these cells and promotes their survivability. The neuritogenic and neuronotrophic effect of gangliosides, their participation in the processes of neurons' regeneration are shown in the in vivo experiments. Gangliosides are carriers of antigenic determinants typical of the cellular surface of neurons (or other cells) as well as of cells of different malignant tumours; typical carcinoembryonal antigenes are revealed among them. Such functions of gangliosides as participation in the processes of intercellular interaction, adhesion, pneuritogenic effect, possible participation to memorize one or another habit are, probably, interrelated and mutually conditioned. Recently the data, being in controversy with the notion that gangliosides are components of receptors of hormones and mediators, are obtained. Evidently, it ought to speak about the modulation of cell response by them on the action of these effectors.     

Integrins and Src: dynamic duo of adhesion signaling

Src family protein tyrosine kinases (SFKs) play important roles downstream of integrin adhesion receptors, and they are necessary for the generation of "outside-in signals" that regulate cytoskeletal organization, cell motility and gene expression in response to cell adhesion. One relatively under-explored facet of this relationship is the possible physical interaction of integrins with SFKs. Recently, it has been established that beta3 integrins and c-Src can interact directly, and this pool of c-Src is activated by cell adhesion to initiate outside-in signaling in platelets, osteoclasts and cells of the vasculature. Here, the biochemical basis for and biological significance of this integrin-SFK interaction is summarized, and I propose a general mechanism for initiation of outside-in integrin signaling.