Role of Cytokine-Induced Glycosylation Changes in Regulating Cell Interactions and Cell Signaling in Inflammatory Diseases and Cancer

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 number of diseases such as cancer and chronic inflammation. In that context, pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases involved in the biosynthesis of carbohydrate chains. These changes in cell surface glycosylation are also known to regulate cell signaling and could contribute to disease pathogenesis. This review summarizes our current knowledge of the glycosylation changes induced by pro-inflammatory cytokines, with a particular focus on cancer and cystic fibrosis, and their consequences on cell interactions and signaling

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

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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

ST6GALNAC5 Expression Decreases the Interactions between Breast Cancer Cells and the Human Blood-Brain Barrier.

International audienceThe ST6GALNAC5 gene that encodes an α2,6-sialyltransferase involved in the biosynthesis of α-series gangliosides, was previously identified as one of the genes that mediate breast cancer metastasis to the brain. We have shown that the expression of ST6GALNAC5 in MDA-MB-231 breast cancer cells resulted in the expression of GD1α ganglioside at the cell surface. By using a human blood-brain barrier in vitro model recently developed, consisting in CD34⁺ derived endothelial cells co-cultivated with pericytes, we show that ST6GALNAC5 expression decreased the interactions between the breast cancer cells and the human blood-brain barrier

Immunocytochemistry and confocal microscopy analysis of gangliosides expression.

<p>MCF-7 cells (<b>A</b>) and Hs578T cells (<b>B</b>) were treated with 40 ng/mL TNF for 48h. Control and treated cells were incubated with anti-G_M3, G_M2, G_D3, G_D2 or G_D1b specific antibodies and revealed with Alexa Fluor^® 488 conjugated anti-mouse IgG or IgM. G_M1a ganglioside was revealed with the FITC-conjugated cholera toxin B subunit from <i>Vibrio cholerae</i>. The nuclei were counter stained with DAPI. Bars: 25 μm. For statistical analysis (<b>C</b>), images of ≥ 100 MCF-7 cells for each condition were collected based on the DAPI signal and were analyzed for G_D3 and G_D2 expression using an automated algorithm on ImageJ software. *: <i>p</i> < 0.05, **: <i>p</i> < 0.01 <i>vs</i>. untreated (control).</p

Primer pairs used for qPCR experiments.

<p>Primer pairs used for qPCR experiments.</p

Mass spectrometry (MALDI-TOF) analysis of permethylated GSL isolated from MCF-7, Hs578T and MDA-MB-231 cells.

<p>Blue circle, Glc; yellow circle, Gal; yellow square, GalNAc; purple diamond, Neu5Ac. Only masses corresponding to N-palmitoyl- (C16:0) or N-lignoceroyl- (C24:0) 2-amino-4-octadecene-1,3-diol (sphingosine) are indicated. Cer*: Ceramide d18:1/C16:0; Cer**: Ceramide d18:1/C24:0.</p

Compositional assignments of singly charged sodiated molecular ions [M + Na]⁺, observed in MALDI-TOF mass spectrometry spectra of permethylated GSL from MCF-7, Hs578T and MDA-MB-231 cells.

<p>Compositional assignments of singly charged sodiated molecular ions [M + Na]⁺, observed in MALDI-TOF mass spectrometry spectra of permethylated GSL from MCF-7, Hs578T and MDA-MB-231 cells.</p

Effect of TNF on GTs expression in MCF-7, Hs578T and MDA-MB-231 cell lines.

<p>Cells were treated with 40 ng/mL TNF for 12-24h. <i>ST3GAL5</i>, <i>ST8SIA1</i>, <i>B4GALNT1</i> and <i>B3GALT4</i> mRNA expression was determined by qPCR. Results were normalized to the expression of HPRT and reported to the expression of GTs in non-treated cells (control). Each bar represents the mean +/- S.E.M. of n ≥ 3 experiments. *: <i>p</i> < 0.05, **: <i>p</i> < 0.01 <i>vs</i>. untreated (control).</p