Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2−) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin β1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin β1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin β1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin β1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin β1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2− cells. All these results suggest that GD2 and integrin β1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

Pharmacokinetics of the disialoganglioside, GD2, a circulating tumor biomarker for neuroblastoma, in nonhuman primates

Background: The ganglioside GD2 is a potential circulating tumor biomarker for the childhood cancer, neuroblastoma. Interpreting the levels of a circulating tumor biomarker depends in part on a knowledge of the biomarker’s clinical pharmacology. Background: The ganglioside GD2 is a potential circulating tumor biomarker for the childhood cancer neuroblastoma. Interpreting the levels of a circulating tumor biomarker depends in part on a knowledge of the biomarker’s clinical pharmacology. Methods: We studied the plasma and cerebrospinal fluid (CSF) pharmacokinetics of the C18 lipoform of GD2 in two nonhuman primates with indwelling subcutaneous CSF lateral ventricular reservoir systems. GD2 was quantified with a validated high-performance liquid chromatography (HPLC)/tandem mass spectrometry assay. GD2 was administered as a short intravenous infusion and frequent plasma and CSF samples were drawn over 72 hours. Results: GD2 plasma concentration declined monoexponentially with a half-life of 16 hours. Clearance was 0.0136 and 0.0131 L/h and volume of distribution (Vd) was 0.035 and 0.038 L/kg in the two animals. Vd was equivalent to plasma volume. Greater than 98% of GD2 in plasma is in a bound form consistent with its known association with lipoproteins and accounting for its limited volume of distribution. GD2 did not cross over from plasma into the CSF. Conclusions: The pharmacokinetic profile of GD2 is favorable for a circulating tumor biomarker. This study demonstrates the value of characterizing the clinical pharmacology of circulating biomarkers to better understand their clinical behavior.

RNA Sequencing-Based Identification of Ganglioside GD2-Positive Cancer Phenotype

The tumor-associated ganglioside GD2 represents an attractive target for cancer immunotherapy. GD2-positive tumors are more responsive to such targeted therapy, and new methods are needed for the screening of GD2 molecular tumor phenotypes. In this work, we built a gene expression-based binary classifier predicting the GD2-positive tumor phenotypes. To this end, we compared RNA sequencing data from human tumor biopsy material from experimental samples and public databases as well as from GD2-positive and GD2-negative cancer cell lines, for expression levels of genes encoding enzymes involved in ganglioside biosynthesis. We identified a 2-gene expression signature combining ganglioside synthase genes ST8SIA1 and B4GALNT1 that serves as a more efficient predictor of GD2-positive phenotype (Matthews Correlation Coefficient (MCC) 0.32, 0.88, and 0.98 in three independent comparisons) compared to the individual ganglioside biosynthesis genes (MCC 0.02–0.32, 0.1–0.75, and 0.04–1 for the same independent comparisons). No individual gene showed a higher MCC score than the expression signature MCC score in two or more comparisons. Our diagnostic approach can hopefully be applied for pan-cancer prediction of GD2 phenotypes using gene expression data.