Profiling of O-acetylated Gangliosides Expressed in Neuroectoderm Derived Cells.

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and OAcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for O-acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of O-acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on O-acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the O-acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among O-acetylated gangliosides, we characterized the expression of OAcGM1, OAcGD3, OAcGD2, OAcGT2, and OAcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the O-acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside O-acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different O-acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of O-acetylated ganglioside profiles in cancer cells.

Identification of 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) as main O-acetylated sialic acid species of GD2 in breast cancer cells.

Mainly restricted to the nervous system in healthy adults, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. Interestingly, O-acetylated forms of GD2, not expressed in human peripheral nerve fibers, are highly expressed in GD2+ tumor cells. Very little information is known regarding the expression of O-acetylated disialogangliosides in breast cancer (BC) cell lines. Here, we analyzed the expression of GD2, GD3 and their O-acetylated forms O-acetyl-GD2 (OAcGD2) and O-acetyl-GD3 (OAcGD3) in BC cells. We used Hs 578T and SUM159PT cell lines, as well as cell clones over-expressing GD3 synthase derived from MDA-MB-231 and MCF-7. Using flow cytometry and immunocytochemistry/confocal microscopy, we report that BC cells express b-series gangliosides GD3 and GD2, as well as significant amounts of OAcGD2. However, OAcGD3 expression was not detected in these cells. O-acetylation of gangliosides isolated from BC cells was examined by LC-MS analysis of sialic acid DMB-derivatives. We report that the main acetylated form of sialic acid expressed in BC gangliosides is 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). These results highlight a close interrelationship between Neu5,9Ac2 and OAcGD2 expression, and suggest that OAcGD2 is synthetized from GD2 and not from OAcGD3 in BC cells.

[Reticular and Golgi glycosylation: Advances and associated diseases]. / Les mécanismes de régulation de la glycosylation - Exemples d'altérations des chaînes glycanniques dans les cancers.

Glycosylation is one of the essential modifications of proteins and lipids. It is carried out mainly in the endoplasmic reticulum and Golgi apparatus, and requires a specific molecular machinery associating several hundreds of glycosyltransferases, glycosidases, transporters and regulating proteins. Modifications of glycosylation are found in numerous diseases, notably in cancers. All types of glycosylation can be affected and this leads to dysfunctions of cellular metabolism. In this review, we present the current knowledge on the regulation of glycosylation mechanisms and illustrate how the alteration of these regulatory mechanisms can lead to abnormal protein and lipid glycosylation, and take part in the development of cancers.

TITLE: Les mécanismes de régulation de la glycosylation - Exemples d'altérations des chaînes glycanniques dans les cancers. ABSTRACT: La glycosylation est l'une des modifications essentielles des protéines et des lipides. Elle s'effectue principalement dans le réticulum endoplasmique et l'appareil de Golgi et fait appel à une machinerie moléculaire spécifique, associant plusieurs centaines de glycosyltransférases, de glycosidases, de transporteurs et de protéines régulatrices. Des modifications de la glycosylation sont retrouvées dans certaines maladies, notamment dans les cancers. Ces altérations peuvent affecter toutes les formes de glycosylation réticulaires et/ou golgiennes, et conduire à des dysfonctionnements du métabolisme cellulaire. Dans cette revue, nous présentons l'état actuel des connaissances des mécanismes de la glycosylation. Nous illustrerons, au travers d'exemples représentatifs, comment l'altération de certains de ces mécanismes de régulation peut affecter les différentes formes de glycosylation des protéines et des lipides et participer au développement des cancers.

Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells.

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.

The EGF Domains of MUC4 Oncomucin Mediate HER2 Binding Affinity and Promote Pancreatic Cancer Cell Tumorigenesis.

The HER2 receptor and its MUC4 mucin partner form an oncogenic complex via an extracellular region of MUC4 encompassing three EGF domains that promotes tumor progression of pancreatic cancer (PC) cells. However, the molecular mechanism of interaction remains poorly understood. Herein, we decipher at the molecular level the role and impact of the MUC4EGF domains in the mediation of the binding affinities with HER2 and the PC cell tumorigenicity. We used an integrative approach combining in vitro bioinformatic, biophysical, biochemical, and biological approaches, as well as an in vivo study on a xenograft model of PC. In this study, we specified the binding mode of MUC4EGF domains with HER2 and demonstrate their "growth factor-like" biological activities in PC cells leading to stimulation of several signaling proteins (mTOR pathway, Akt, and ß-catenin) contributing to PC progression. Molecular dynamics simulations of the MUC4EGF/HER2 complexes led to 3D homology models and identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results will pave the way to the design of potential MUC4/HER2 inhibitors targeting the EGF domains of MUC4. This strategy will represent a new efficient alternative to treat cancers associated with MUC4/HER2 overexpression and HER2-targeted therapy failure as a new adapted treatment to patients.

O-acetylated Gangliosides as Targets for Cancer Immunotherapy.

O-acetylation of sialic acid residues is one of the main modifications of gangliosides, and modulates ganglioside functions. O-acetylation of gangliosides is dependent on sialyl-O-acetyltransferases and sialyl-O-acetyl-esterase activities. CAS1 Domain-Containing Protein 1 (CASD1) is the only human sialyl-O-acetyltransferases (SOAT) described until now. O-acetylated ganglioside species are mainly expressed during embryonic development and in the central nervous system in healthy adults, but are re-expressed during cancer development and are considered as markers of cancers of neuroectodermal origin. However, the specific biological roles of O-acetylated gangliosides in developing and malignant tissues have not been extensively studied, mostly because of the requirement of specific approaches and tools for sample preparation and analysis. In this review, we summarize our current knowledge of ganglioside biosynthesis and expression in normal and pathological conditions, of ganglioside O-acetylation analysis and expression in cancers, and of the possible use of O-acetylated gangliosides as targets for cancer immunotherapy.

Glycosylation changes in inflammatory diseases.

Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in a number of inflammatory diseases. Pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of glycan chains, inducing the expression of specific carbohydrate antigens at the cell surface that can be recognized by different types of lectins or by bacterial adhesins, contributing to the development of diseases. Glycosylation can also regulate biological functions of immune cells by recruiting leukocytes to inflammation sites with pro- or anti-inflammatory effects. Cell surface proteoglycans provide a large panel of binding sites for many mediators of inflammation, and regulate their bio-availability and functions. In this review, we summarize the current knowledge of the glycosylation changes occurring in mucin type O-linked glycans, glycosaminoglycans, as well as in glycosphingolipids, with a particular focus on cystic fibrosis and neurodegenerative diseases, and their consequences on cell interactions and disease progression.

Gangliosides: The Double-Edge Sword of Neuro-Ectodermal Derived Tumors.

Gangliosides, the glycosphingolipids carrying one or several sialic acid residues, are mostly localized at the plasma membrane in lipid raft domains and implicated in many cellular signaling pathways mostly by interacting with tyrosine kinase receptors. Gangliosides are divided into four series according to the number of sialic acid residues, which can be also modified by O-acetylation. Both ganglioside expression and sialic acid modifications can be modified in pathological conditions such as cancer, which can induce either pro-cancerous or anti-cancerous effects. In this review, we summarize the specific functions of gangliosides in neuro-ectodermal derived tumors, and their roles in reprogramming the lipidomic profile of cell membrane occurring with the induction of epithelial-mesenchymal transition.

TNF differentially regulates ganglioside biosynthesis and expression in breast cancer cell lines.

Gangliosides are glycosphingolipids concentrated in glycolipid-enriched membrane microdomains. Mainly restricted to the nervous system in healthy adult, complex gangliosides such as GD3 and GD2 have been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. GD3 synthase (GD3S), the key enzyme that controls the biosynthesis of complex gangliosides, was shown to be over-expressed in Estrogen Receptor (ER)-negative breast cancer tumors, and associated with a decreased overall survival of patients. We previously demonstrated that GD3S expression in ER-negative breast cancer cells induced a proliferative phenotype and an increased tumor growth. In addition, our results clearly indicate that Tumor Necrosis Factor (TNF) induced GD3S over-expression in breast cancer cells via NFκB pathway. In this study, we analyzed the effect of TNF on ganglioside biosynthesis and expression in breast cancer cells from different molecular subtypes. We showed that TNF up-regulated the expression of GD3S in MCF-7 and Hs578T cells, whereas no change was observed for MDA-MB-231. We also showed that TNF induced an increased expression of complex gangliosides at the cell surface of a small proportion of MCF-7 cells. These results demonstrate that TNF differentially regulates gangliosides expression in breast cancer cell lines and establish a possible link between inflammation at the tumor site environment, expression of complex gangliosides and tumor development.