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

Farhana Yesmin,Koichi Furukawa,Yuki Ohkawa,Yuhsuke Ohmi,Kei Kaneko,Robiul H Bhuiyan,Orie Tajima,Pu Zhang,Nai-Kong V Cheung,Norihiro Kotani,Tetsuya Okajima,Keiko Furukawa,Kazunori Hamamura,Koichi Honke,Satoko Yamamoto,Mariko Kambe

doi:10.3390/ijms23010423

Abstract

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.

Highlights

Gangliosides are sialic acid-containing glycosphingolipids, and they are expressed in almost all the cells and tissues of vertebrates [1]
We identify the membrane molecules interacting with ganglioside GD2 on the surface of human melanoma cells using EMARS/MS, and integrins were identified as representative molecules to associate with GD2
The detection of GD2 by monoclonal antibodies (mAbs) 3F8 is shown in Supplementary Figure S1

Summary

Introduction

Gangliosides are sialic acid-containing glycosphingolipids, and they are expressed in almost all the cells and tissues of vertebrates [1]. Complex gangliosides are commonly enriched in the nervous tissues of many animals in common, and have been considered to play important roles in the development and function of the nervous system [2]. Some gangliosides were reported to be expressed in particular cancer cells and tissues, and so they have been considered to be cancer-associated carbohydrate antigens [3,4]. Since the cDNAs of ganglioside synthetic enzymes were isolated, it became possible to investigate the roles of gangliosides in various cells and tissues [8]. The genetic engineering of glycosyltransferase genes in cultured cells and experimental animals have enabled us to clarify significant roles of gangliosides, and their mechanisms in development and carcinogenesis [9]

Methods

Results

Conclusion