Study of the role of the GD3 synthase and complex gangliosides in the proliferation and the migration of the breast cancer cells

Le rôle des gangliosides complexes di- ou trisialylés (GD3, GD2, GT3) dans le développement des cancers d’origine neuro-ectodermique (mélanome, neuroblastome) a été clairement démontré. Ces gangliosides sont également surexprimés dans 50% des carcinomes mammaires canalaires infiltrants et l’expression de la GD3 synthétase (GD3S) contrôlant leur biosynthèse, est augmentée dans les tumeurs de sein ER-négatives, avec une diminution de la survie des patientes. Notre laboratoire a montré que l’expression de la GD3S dans les cellules cancéreuses de sein MDA-MB-231 induit une augmentation de la migration et de la prolifération en conditions de sevrage, liée à une activation constitutive du récepteur c-Met et des voies de signalisation intracellulaires Erk/MAPK et PI3K/Akt par le GD2. Nous avons développé un autre modèle cellulaire dérivant des cellules cancéreuses mammaires MCF-7 sur-exprimant la GD3S. Ces cellules accumulent principalement les gangliosides GD1b et GT3. Nous avons pu montrer une augmentation de la migration mais aucun effet sur la prolifération n’a été observé. L’analyse en spectrométrie de masse des glycosphingolipides a montré que les MCF-7 expriment des quantités élevées de globosides et de faibles quantités de gangliosides par rapport aux cellules MDA-MB-231. Dans les cellules MCF7 GD3S+, nous avons démontré que la GD3S était capable de synthétiser du GT3 à partir du GD3, mais également des gangliosides inhabituels tétra- et penta-sialylés. Nos résultats montrent que les cellules cancéreuses mammaires présentent des profils gangliosidiques différents et que la modification du phénotype cellulaire induite par l'expression de la GD3S dépend du type cellulaire.The role of di- and trisialylated complex gangliosides (GD3, GD2 and GT3) in the development of neuro-ectoderm derived cancers (melanoma, neuroblastoma) has been clearly demonstrated. Complex gangliosides are also over-expressed in 50% of breast invasive ductal carcinomas and the expression of the GD3 synthase (GD3S) that controls their biosynthesis, is increased in ER-negatives breast cancer tumors, associated with a decreased overall survival of the patients. Our lab has previously demonstrated that GD3S expression in MDA-MB-231 breast cancer cells induced an increase of the cell migration and proliferation in serum-free conditions, associated to a constitutive GD2-dependent constitutive activation of c-Met receptor and Erk/MAPK et PI3K/Akt signaling pathways. We have developed another cellular model deriving from MCF-7 breast cancer cells, over-expressing the GD3S. These cells mainly express GD1b and GT3 gangliosides. We have shown the increased migration of MCF7 GD3S+, but no effect on the proliferation was observed. Mass spectrometry analysis of total glycosphingolipids has shown that MCF-7 cells express a high level of globosides and a low level of gangliosides compared to MDA-MB-231 cells. In MCF7 GD3S+ cells, we demonstrated that the GD3S was able to synthesize GT3 from GD3, but also other unusual tetra- and penta-sialylated gangliosides. Our results show that breast cancer cell lines express different gangliosidic profiles and that the modification of the cellular phenotype resulting from the expression of the GD3S is depending on the cell type

Etude du rôle de la GD3 synthétase et des gangliosides complexes dans la prolifération et la migration des cellules de cancer du sein

Le rôle des gangliosides complexes di- ou trisialylés (GD3, GD2, GT3) dans le développement des cancers d origine neuro-ectodermique (mélanome, neuroblastome) a été clairement démontré. Ces gangliosides sont également surexprimés dans 50% des carcinomes mammaires canalaires infiltrants et l expression de la GD3 synthétase (GD3S) contrôlant leur biosynthèse, est augmentée dans les tumeurs de sein ER-négatives, avec une diminution de la survie des patientes. Notre laboratoire a montré que l expression de la GD3S dans les cellules cancéreuses de sein MDA-MB-231 induit une augmentation de la migration et de la prolifération en conditions de sevrage, liée à une activation constitutive du récepteur c-Met et des voies de signalisation intracellulaires Erk/MAPK et PI3K/Akt par le GD2. Nous avons développé un autre modèle cellulaire dérivant des cellules cancéreuses mammaires MCF-7 sur-exprimant la GD3S. Ces cellules accumulent principalement les gangliosides GD1b et GT3. Nous avons pu montrer une augmentation de la migration mais aucun effet sur la prolifération n a été observé. L analyse en spectrométrie de masse des glycosphingolipides a montré que les MCF-7 expriment des quantités élevées de globosides et de faibles quantités de gangliosides par rapport aux cellules MDA-MB-231. Dans les cellules MCF7 GD3S+, nous avons démontré que la GD3S était capable de synthétiser du GT3 à partir du GD3, mais également des gangliosides inhabituels tétra- et penta-sialylés. Nos résultats montrent que les cellules cancéreuses mammaires présentent des profils gangliosidiques différents et que la modification du phénotype cellulaire induite par l'expression de la GD3S dépend du type cellulaire.The role of di- and trisialylated complex gangliosides (GD3, GD2 and GT3) in the development of neuro-ectoderm derived cancers (melanoma, neuroblastoma) has been clearly demonstrated. Complex gangliosides are also over-expressed in 50% of breast invasive ductal carcinomas and the expression of the GD3 synthase (GD3S) that controls their biosynthesis, is increased in ER-negatives breast cancer tumors, associated with a decreased overall survival of the patients. Our lab has previously demonstrated that GD3S expression in MDA-MB-231 breast cancer cells induced an increase of the cell migration and proliferation in serum-free conditions, associated to a constitutive GD2-dependent constitutive activation of c-Met receptor and Erk/MAPK et PI3K/Akt signaling pathways. We have developed another cellular model deriving from MCF-7 breast cancer cells, over-expressing the GD3S. These cells mainly express GD1b and GT3 gangliosides. We have shown the increased migration of MCF7 GD3S+, but no effect on the proliferation was observed. Mass spectrometry analysis of total glycosphingolipids has shown that MCF-7 cells express a high level of globosides and a low level of gangliosides compared to MDA-MB-231 cells. In MCF7 GD3S+ cells, we demonstrated that the GD3S was able to synthesize GT3 from GD3, but also other unusual tetra- and penta-sialylated gangliosides. Our results show that breast cancer cell lines express different gangliosidic profiles and that the modification of the cellular phenotype resulting from the expression of the GD3S is depending on the cell type.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

How Do Gangliosides Regulate RTKs Signaling?

Gangliosides, the glycosphingolipids carrying one or several sialic acid residues, are located on the outer leaflet of the plasma membrane in glycolipid-enriched microdomains, where they interact with molecules of signal transduction pathways including receptors tyrosine kinases (RTKs). The role of gangliosides in the regulation of signal transduction has been reported in many cases and in a large number of cell types. In this review, we summarize the current knowledge on the biosynthesis of gangliosides and the mechanism by which they regulate RTKs signaling

Estradiol represses the G(D3) synthase gene ST8SIA1 expression in human breast cancer cells by preventing NFκB binding to ST8SIA1 promoter.

Recent data have underlined a possible role of G(D3) synthase (GD3S) and complex gangliosides in Estrogen Receptor (ER) negative breast cancer progression. Here, we describe the main transcript of the GD3S coding gene ST8SIA1 expressed in breast tumors. We characterized the corresponding core promoter in Hs578T breast cancer cells and showed that estradiol decreases ST8SIA1 mRNA expression in ER-positive MCF-7 cells and ERα-transfected ER-negative Hs578T cells. The activity of the core promoter sequence of ST8SIA1 is also repressed by estradiol. The core promoter of ST8SIA1 contains two putative Estrogen Response Elements (ERE) that were not found to be involved in the promoter activity pathway. However, NFκB was shown to be involved in ST8SIA1 transcriptional activation and we demonstrated that estradiol prevents NFκB to bind to ST8SIA1 core promoter in ERα expressing breast cancer cells by inhibiting p65 and p50 nucleus localization. The activation of NFκB pathway in ER-negative tumors, due to the absence of estradiol signaling, might explain the overexpression of G(D3) synthase in this tumor subtype

Recombinant fungal lectin as a new tool to investigate O-GlcNAcylation processes

International audienceGlycosylation is a group of post-translational modifications that displays a large variety of structures and are implicated in a plethora of biological processes. Therefore, studying glycosylation requires different technical approaches and reliable tools, lectins being part of them. Here we describe the use of the recombinant mushroom lectin PVL to discriminate O-GlcNAcylation, a modification consisting in the attachment of a single N-acetylglucosamine residue to proteins confined within the cytosolic, nuclear and mitochondrial compartments. rPVL displays significantly stronger affinity for GlcNAc over Neu5Ac residues as verified by thermal shift assays and surface plasmon resonance experiments, being therefore an excellent alternative to WGA. Labelling of rPVL with biotin or HRP (horseradish peroxidase) allows its useful and efficient utilization by Western blot. The staining of whole cell lysates with labelled-rPVL was dramatically decreased in response to OGT (O-GlcNAc transferase) knockdown and seen to increase after pharmacological blockade of OGA (O-GlcNAcase). Also, HRP-rPVL seemed to be more sensitive than the anti-O-GlcNAc antibody RL2. Thus, rPVL is a potent new tool to selectively detect O-GlcNAcylated proteins

α-L-fucosidase inhibition by pyrrolidine-ferrocene hybrids: rationalization of ligand-binding properties by structural studies.

International audienceEnhanced metabolism of fucose through fucosidase overexpression is a signature of some cancer types, thus suggesting that fucosidase-targetted ligands could play the role of drug-delivery vectors. Herein, we describe the synthesis of a new series of pyrrolidine-ferrocene conjugates, consisting of a L-fuco-configured dihydroxypyrrolidine as the fucosidase ligand armed with a cytotoxic ferrocenylamine moeity. Three-dimensional structures of several of these fucosidase inhibitors reveal transition-state-mimicking (3)E conformations. Elaboration with the ferrocenyl moiety results in sub-micromolar inhibitors of both bovine and bacterial fucosidases, with the 3D structure of the latter revealing electron density indicative of highly mobile alkylferrocene compounds. The best compounds show a strong antiproliferative effect, with up to 100% inhibition of the proliferation of MDA-MB-231 cancer cells at 50 μM

Accumulation of Unusual Gangliosides GQ3 and GP3 in Breast Cancer Cells Expressing the GD3 Synthase

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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

Identification of human GD3S transcripts in breast cancer tumors and Hs578T cells.

<p>(A) Schematic representation of the 5′-RACE strategy. First strand cDNA synthesis was performed with a <i>ST8SIA1</i> specific primer. cDNA was dC-tailed at 3′end and amplified by PCR using Anchor Primer and GSP1. Nested PCR was performed using AUAP and GSP2 primers. (B) Schematic representation of the main 5′-ends of GD3S transcripts expressed in Hs578T cells and breast cancer tumor samples (#137, #142 and #148). The size of intronic sequences between E2 and the different first exon are shown. Position of PCR primers used for specific amplification of T1 transcript is indicated by black arrows. (C) qPCR analysis of T1 transcript (grey) and total <i>ST8SIA1</i> (black) expression, related to <i>HPRT</i>, in 20 ER-negative IDC samples and 2 breast cancer cell lines (Hs578T and MCF-7).</p