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Gangliosides, sialic acid-containing glycosphingolipids, are widely involved in regulations of signal transductions to control cellular functions. It has been suggested that GM3, the simplest structure among gangliosides, is involved in insulin resistance, whereas it remains unclear whether insulin signaling diminished by GM3 actually aggravates the pathological conditions in metabolic disorders. Moreover, the functional roles of gangliosides in the regulation of insulin signaling have not yet been fully elucidated in liver or hepatocytes despite that it is one of the major insulin-sensitive organs. To understand physiological roles of GM3 in metabolic homeostasis in liver, we conducted a high fat diet (HFD) loading experiment using double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase, which lack all gangliosides except GM3, as well as wild-type (WT) mice. DKO mice were strikingly resistant to HFD-induced hepatosteatosis, and hepatic lipogenesis-related molecules including insulin signaling components were down-regulated in HFD-fed DKO. Furthermore, we established primary hepatocyte cultures from DKO and WT mice, and examined their responses to insulin in vitro . Following insulin stimulation, DKO hepatocytes expressing GM3 showed attenuated expression and/or activations in the downstream components compared with WT hepatocytes expressing GM2. While insulin stimulation induced lipogenic proteins in hepatocytes from both genotypes, their expression levels were lower in DKO than in WT hepatocytes after insulin treatment. All our findings suggest that the modified gangliosides, i.e., a shift to GM3 from GM2, might exert a suppressive effect on lipogenesis by attenuating insulin signaling at least in mouse hepatocytes, which might result in protection of HFD-induced hepatosteatosis.

Ganglioside GD3 is widely expressed in human malignant melanomas, and has been reported to be involved in the increased cell proliferation and invasion. In this study, we established GM3-, GM2-, GM1-, GD3-, or GD2-expressing melanoma cell lines by transfecting cDNAs of glyscosyltransferases, and effects of individual gangliosides on the cell phenotypes and signals were examined. The phenotypes of established ganglioside-expressing cells were quite different, i.e. cell growth increased as following order; GD2+, GD3+ > GM1+, GM2+, GM3+ cells. Cell invasion activity increased as GD3+ ≧ GM2+ > GM1+, GM3+, GD2+ cells. Intensity of cell adhesion to collagen I (CL-I) and spreading increased as GD2+ >> GD3+, GM1+ > GM2+, GM3+ cells. In particular, cell adhesion of GD2+ cells was markedly strong. As for cell migration velocity, GD2+ cells were slower than all other cells. The immunocytostaining revealed close localization of gangliosides and F-actin in lamellipodia. Immunoblotting of phosphorylated p130Cas and paxillin by serum treatment reveled that these phosphorylations were more increased in GD3+ cells than in GD2+ or GM3+ cells, while phosphorylation of Akt underwent similarly increased phosphorylation between GD3+ and GD2+ cells compared with GM3+ cells. While GD2 and GD3 enhanced cell growth, GD3 might also contribute in cell invasion. On the other hand, GD2 might contribute in the solid fixation of melanoma cells at metastasized sites. These results suggested that individual gangliosides exert distinct roles in the different aspects of melanomas by differentially regulating cytoskeletons and signaling molecules.

While various glycosphingolipids were identified as cancer‐associated carbohydrate antigens to be used as tumor markers, disialylated gangliosides such as GD3 and GD2 have particularly attracted attention from many researchers as promising cancer‐associated antigens. Simultaneously, their functions in cancer and normal tissues have also been reported. Although GD3 is expressed at the early neural developmental stage and in various cancers, it is also found in the activated status of some normal cells such as astrocytes and lymphocytes. On the other hand, GD2 is expressed in more restricted cells than GD3, enabling anti‐GD2 immune therapy to be more applicable for immunotherapy. Recently, the expression of GD2 has been reported in various epithelial cancers and neuroectoderm‐derived tumors. The involvement of GD2 in cancer stem cell propertiesand the roles of GD2 in the signal modulation to bring about cancer stemness are now some of the most fascinating research topics. Cancer immunotherapy targeting GD2 by anti‐GD2 antibody or anti‐GD2 CAR‐T is now widely being challenged with various modifications such as combination with cytokines, chemotherapy, or immune checkpoint blocking. Among cancer‐associated antigens, ganglioside GD2 is the most crucial and essential carbohydrate antigen as a modulator of malignant signals and as a target of immunotherapy for refractory cancers. In particular, GD2 is now believed to be a marker of cancer stem cells and to be essentially involved in the features of cancer stemness.

Cancer‐associated glycosphingolipids have been used as markers for diagnosis and targets for immunotherapy of malignant tumors. Recent progress in the analysis of their implications in the malignant properties of cancer cells revealed that cancer‐associated glycosphingolipids are not only tumor markers, but also functional molecules regulating various signals introduced by membrane microdomains, lipid rafts. In particular, a novel approach, enzyme‐mediated activation of radical sources combined with mass spectrometry, has enabled us to clarify the mechanisms by which cancer‐associated glycosphingolipids regulate cell signals based on the interaction with membrane molecules and formation of molecular complexes on the cell surface. Novel findings obtained from these approaches are now providing us with insights into the development of new anticancer therapies targeting membrane molecular complexes consisting of cancer‐associated glycolipids and their associated membrane molecules. Thus, a new era of cancer‐associated glycosphingolipids has now begun. Cancer‐associated glycosphingolipids have been used as markers for diagnosis and targets for immunotherapy of malignant tumors. Recent studies have revealed that they are not only tumor markers, but also functional molecules regulating various signals at lipid rafts. In particular, a novel approach, enzyme‐mediated activation of radical sources combined with mass spectrometry, has enabled us to clarify the mechanisms by which cancer‐associated glycosphingolipids regulate cell signals, leading to development of novel therapies targeting membrane molecular complexes consisting of cancer‐associated glycolipids and their associating membrane molecules.

Immunotherapy of malignant cancers is now becoming one of representative approaches to overcome cancers. To construct strategies for immunotherapy, presence of tumor-specific antigens should be a major promise. A number of cancer specific- or cancer-associated antigens have been reported based on various experimental sets and various animal systems. The most reasonable strategy to define tumor-specific antigens might be “autologous typing” performed by Old’s group, proposing three classes of tumor-antigens recognized by host immune systems of cancer patients. Namely, class 1, individual antigens that is present only in the patient’s sample analyzed; class 2, shared antigens that can be found only in some group of cancers in some patients, but not in normal cells and tissues; class 3, universal antigens that are present in some cancers but also in normal cells and tissues with different densities. Sen Hakomori reported there were novel carbohydrates in cancers that could not be detected in normal cells mainly by biochemical approaches. Consequently, many of class 2 cancer-specific antigens have been revealed to be carbohydrate antigens, and been used for cancer diagnosis and treatment. Not only as cancer markers, but roles of those cancer-associated carbohydrates have also been recognized as functional molecules in cancer cells. In particular, roles of complex carbohydrates in the regulation of cell signaling on the cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been reported by Hakomori and many other researchers including us. The processes and present status of these studies on cancer-associated glycolipids were summarized.

Exosomes (small extracellular vesicles: EVs) have attracted increasing attention from basic scientists and clinicians since they play important roles in cell-to-cell communication in various biological processes. Various features of EVs have been elucidated regarding their contents, generation and secretion mechanisms, and functions in inflammation, regeneration, and cancers. These vesicles are reported to contain proteins, RNAs, microRNAs, DNAs, and lipids. Although the roles of individual components have been rigorously studied, the presence and roles of glycans in EVs have rarely been reported. In particular, glycosphingolipids in EVs have not been investigated to date. In this study, the expression and function of a representative cancer-associated ganglioside, GD2, in malignant melanomas was investigated. Generally, cancer-associated gangliosides have been shown to enhance malignant properties and signals in cancers. Notably, EVs derived from GD2-expressing melanomas enhanced the malignant phenotypes of GD2-negative melanomas, such as cell growth, invasion, and cell adhesion, in a dose-dependent manner. The EVs also induced increased phosphorylation of signaling molecules such as EGF receptor and focal adhesion kinase. These results suggest that EVs released from cancer-associated ganglioside-expressing cells exert many functions that have been reported as a function of these gangliosides and regulate microenvironments, including total aggravation of heterogeneous cancer tissues, leading to more malignant and advanced cancer types.

Extracellular vesicles (EVs) play important roles in intercellular communication in various biological events. In particular, EVs released from cancer cells have attracted special attention. Although it has been reported that cancer-associated glycosphingolipids play important roles in the enhancement of malignant properties of cancer cells, the presence, behavior, and roles of glycosphingolipids in EVs have not been elucidated. Recently, we reported crucial roles of EVs expressing gangliosides, GD2, and/or GD3 in the enhancement of cancer properties in malignant melanomas and gliomas. However, how EVs containing cancer-associated glycosphingolipids play their roles has not been reported to date. Here, we studied spatio-temporal mechanisms for GD3/GD2-containing EVs released from gliomas in the actions toward target cells. Proteome analyses of EVs with/without GD3/GD2 revealed an equally high concentration of integrin isoforms in both GD3/GD2+ and GD3/GD2- EVs. PKH26-labeled EVs attached, invaded, and distributed to/in the target cells within 1 h. GD3/GD2 formed molecular complexes with integrins on EVs as elucidated by immunoprecipitation/immunoblotting and immunocytostaining. The addition of antibodies reactive with GD3, GD2, or integrins resulted in the suppression of the enhancing effects of EVs in the cell adhesion assay. The addition of GD3/GD2 + EVs to GD3/GD2- cells clearly increased the phosphorylation levels of the PDGF receptor, FAK, and Erk1/2 in immunoblotting, suggesting GD3/GD2+ EVs activate the signaling pathway in the target cells within 15 min after addition. Anti-ganglioside antibodies clearly blocked signaling with EVs. In conclusion, EVs released from GD3/GD2-expressing glioma cells enhance cancer phenotypes and malignant signals via the cluster formation of integrins and GD3/GD2 on EVs, leading to the regulation of the cancer microenvironment.

Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity. In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL‐I). Although stimulation of melanoma N1 cells (GD3+ and GD3−) with either HGF or adhesion to CL‐I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells. Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells. When resistance to induced apoptosis by H2O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL‐I showed clearly low percentages of dead cells compared with GD3− cells or GD3+ cells treated with either one of the stimulants. Cell growth measured by 5‐ethynyl‐2‘ deoxyuridine uptake also showed synergistic effects in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas. Ganglioside GD3 has been considered to be a melanoma‐associated glycolipid antigen. Here, we demonstrated that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas.

Since globotetraosylceramide was defined as a major glycosphingolipid in human erythrocytes, various glycolipids have been found in normal cells and diseased organs. However, the implications of their polymorphic structures in the function of individual cells and tissues have not been clarified. Genetic manipulation of glycosphingolipids in cultured cells and experimental animals has enabled us to substantially elucidate their roles. In fact, great progress has been achieved in the last 70 years in revealing that glycolipids are essential in the maintenance of integrity of nervous tissues and other organs. Furthermore, the correct composition of glycosphingolipids has been shown to be critical for the protection against inflammation and degeneration. Here, we summarized historic information and current knowledge about glycosphingolipids, with a focus on their involvement in inflammation and degeneration. This topic is significant for understanding the biological responses to various stresses, because glycosphingolipids play roles in the interaction with various intrinsic and extrinsic factors. These findings are also important for the application of therapeutic interventions of various diseases.

Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Recently, novel roles of gangliosides in the regulation of complement system were reported. Here we summarized roles of gangliosides in the formation and maintenance of membrane microdomains in brain tissues by comparing complement activation, inflammatory reaction and disruption of glycolipid-enriched microdomain (GEM)/rafts among several mutant mice of ganglioside synthases. Depending on the defects in ganglioside compositions, corresponding up-regulation of complement-related genes, proliferation of astrocytes and infiltration of microglia were found with gradual severity. Immunoblotting of fractions separated by sucrose density gradient ultracentrifugation revealed that DAF and NCAM having GPI-anchors tended to disappear from the raft fraction with intensities of DKO > GM2/GD2 synthase KO > GD3 synthase KO > WT. The lipid raft markers tended to disperse from the raft fractions with similar intensities. Phospholipids and cholesterol also tended to decrease in GEM/rafts in GM2/GD2 synthase KO and DKO, although total amounts were almost equivalent. All these results indicate that GEM/rafts architecture is destroyed by ganglioside deficiency with gradual intensity depending on the degree of defects of their compositions. Implication of inflammation caused by deficiency of gangliosides in various neurodegenerative diseases was discussed.