Cancer-Associated Glycosphingolipids as Tumor Markers and Targets for Cancer Immunotherapy.

Aberrant expression of glycosphingolipids is a hallmark of cancer cells and is associated with their malignant properties. Disialylated gangliosides GD2 and GD3 are considered as markers of neuroectoderm origin in tumors, whereas fucosyl-GM1 is expressed in very few normal tissues but overexpressed in a variety of cancers, especially in small cell lung carcinoma. These gangliosides are absent in most normal adult tissues, making them targets of interest in immuno-oncology. Passive and active immunotherapy strategies have been developed, and have shown promising results in clinical trials. In this review, we summarized the current knowledge on GD2, GD3, and fucosyl-GM1 expression in health and cancer, their biosynthesis pathways in the Golgi apparatus, and their biological roles. We described how their overexpression can affect intracellular signaling pathways, increasing the malignant phenotypes of cancer cells, including their metastatic potential and invasiveness. Finally, the different strategies used to target these tumor-associated gangliosides for immunotherapy were discussed, including the use and development of monoclonal antibodies, vaccines, immune system modulators, and immune effector-cell therapy, with a special focus on adoptive cellular therapy with T cells engineered to express chimeric antigen receptors.

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.

Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation.

The mammalian mono-α2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 O-glycans leading to the formation of di-sialylated O-glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the ST8SIA6 gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish α2,8-sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of α2,8-sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.

TNF up-regulates ST3GAL4 and sialyl-Lewisx expression in lung epithelial cells through an intronic ATF2-responsive element.

We have previously shown that tumor necrosis factor (TNF) induced the up-regulation of the sialyltransferase gene ST3GAL4 (α2,3-sialyltransferase gene) BX transcript through mitogen- and stress-activated kinase 1/2 (MSK1/2), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. This up-regulation resulted in sialyl-Lewisx (sLex) overexpression on high-molecular-weight glycoproteins in inflamed airway epithelium and increased the adhesion of Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells. In the present study, we describe a TNF-responsive element in an intronic region of the ST3GAL4 gene, whose TNF-dependent activity is repressed by ERK/p38 and MSK1/2 inhibitors. This TNF-responsive element contains potential binding sites for ETS1 and ATF2 transcription factors related to TNF signaling. We also show that ATF2 is involved in TNF responsiveness, as well as in TNF-induced ST3GAL4 BX transcript and sLex overexpression in A549 lung epithelial cells. Moreover, we show that TNF induces the binding of ATF2 to the TNF-responsive element. Altogether, these data suggest that ATF2 could be a potential target to prevent inflammation-induced P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.

Diverse subcellular localizations of the insect CMP-sialic acid synthetases.

The occurrence and biological importance of sialic acid (Sia) and its metabolic enzymes in insects have been studied using Drosophila melanogaster. The most prominent feature of D. melanogaster CMP-Sia synthetase (DmCSS) is its Golgi-localization, contrasted with nuclear localization of vertebrate CSSs. However, it remains unclear if the Golgi-localization is common to other insect CSSs and why it happens. To answer these questions, Aedes aegypti (mosquito) CSS (AaCSS) and Tribolium castaneum (beetle) CSS (TcCSS) were cloned and characterized for their activity and subcellular localization. Our new findings show: (1) AaCSS and TcCSS share a common overall structure with DmCSS in terms of evolutionarily conserved motifs and the absence of the C-terminal domain typical to vertebrate CSSs; (2) when expressed in mammalian and insect cells, AaCSS and TcCSS showed in vivo and in vitro CSS activities, similar to DmCSS. In contrast, when expressed in bacteria, they lacked CSS activity because the N-terminal hydrophobic region appeared to induce protein aggregation; (3) when expressed in Drosophila S2 cells, AaCSS and TcCSS were predominantly localized in the ER, but not in the Golgi. Surprisingly, DmCSS was mainly secreted into the culture medium, although partially detected in Golgi. Consistent with these results, the N-terminal hydrophobic regions of AaCSS and TcCSS functioned as a signal peptide to render them soluble in the ER, while the N-terminus of DmCSS functioned as a membrane-spanning region of type II transmembrane proteins whose cytosolic KLK sequence functioned as an ER export signal. Accordingly, the differential subcellular localization of insect CSSs are distinctively more diverse than previously recognized.

Gangliosides: Structures, Biosynthesis, Analysis, and Roles in Cancer.

Gangliosides are acidic glycosphingolipids containing one or more sialic acid residues. They are essential compounds at the outer leaflet of the plasma membrane, where they interact with phospholipids, cholesterol, and transmembrane proteins, forming lipid rafts. They are involved in cell adhesion, proliferation, and recognition processes, as well as in the modulation of signal transduction pathways. These functions are mainly governed by the glycan moiety, and changes in the structures of gangliosides occur under pathological conditions, particularly in neuro-ectoderm-derived cancers. With the progress in mass spectrometry analysis of gangliosides, their role in cancer progression can be now investigated in more detail. In this review we summarize the current knowledge on the biosynthesis of gangliosides and their role in cancers, together with the recent development of cancer immunotherapy targeting gangliosides.

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

The 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.

TNF induces the expression of the sialyltransferase ST3Gal IV in human bronchial mucosa via MSK1/2 protein kinases and increases FliD/sialyl-Lewis(x)-mediated adhesion of Pseudomonas aeruginosa.

We have shown previously that the pro-inflammatory cytokine TNF (tumour necrosis factor) could drive sLe(x) (sialyl-Lewis(x)) biosynthesis through the up-regulation of the BX transcript isoform of the ST3GAL4 (ST3 ß-galactoside α-2,3-sialyltransferase 4) sialyltransferase gene in lung epithelial cells and human bronchial mucosa. In the present study, we show that the TNF-induced up-regulation of the ST3GAL4 BX transcript is mediated by MSK1/2 (mitogen- and stress-activated kinase 1/2) through the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and increases sLe(x) expression on high-molecular-mass glycoproteins in inflamed airway epithelium. We also show that the TNF-induced sLe(x) expression increases the adhesion of the Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells in a FliD-dependent manner. These results suggest that ERK and p38 MAPK, and the downstream kinase MSK1/2, should be considered as potential targets to hamper inflammation, bronchial mucin glycosylation changes and P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.

Accumulation of GD1α Ganglioside in MDA-MB-231 Breast Cancer Cells Expressing ST6GalNAc V.

α-Series gangliosides define a particular sub-class of glycosphingolipids containing sialic acid α2,6-linked to GalNAc residue that was isolated as a minor compound from the brain. The sialyltransferase ST6GalNAc V was cloned from mouse brain and showed α2,6-sialyltransferase activity almost exclusively for GM1b, to form GD1α and is considered as the main enzyme involved in the biosynthesis of α-series gangliosides. Recently, ST6GALNAC5 was identified as one of the genes over-expressed in breast cancer cell populations selected for their ability to produce brain metastasis. However, the capacity of human breast cancer cells to produce α-series gangliosides has never been clearly demonstrated. Here, we show by stable transfection and MS-MS analysis of total glycosphingolipids that ST6GALNAC5 expressing MDA-MB-231 breast cancer cells accumulate GD1α ganglioside (IV3Neu5Ac1, III6Neu5Ac1Gg4-Cer).

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

Enhanced 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.

The ganglioside G(D2) induces the constitutive activation of c-Met in MDA-MB-231 breast cancer cells expressing the G(D3) synthase.

We have recently established and characterized cellular clones deriving from MDA-MB-231 breast cancer cells that express the human G(D3) synthase (GD3S), the enzyme that controls the biosynthesis of b- and c-series gangliosides. The GD3S positive clones show a proliferative phenotype in the absence of serum or growth factors and an increased tumor growth in severe immunodeficient mice. This phenotype results from the constitutive activation of the receptor tyrosine kinase c-Met in spite of the absence of ligand and subsequent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/Akt pathways. Here, we show by mass spectrometry analysis of total glycosphingolipids that G(D3) and G(D2) are the main gangliosides expressed by the GD3S positive clones. Moreover, G(D2) colocalized with c-Met at the plasma membrane and small interfering RNA silencing of the G(M2)/G(D2) synthase efficiently reduced the expression of G(D2) as well as c-Met phosphorylation and reversed the proliferative phenotype. Competition assays using anti-G(D2) monoclonal antibodies also inhibit proliferation and c-Met phosphorylation of GD3S positive clones in serum-free conditions. Altogether, these results demonstrate the involvement of the disialoganglioside G(D2) in MDA-MB-231 cell proliferation via the constitutive activation of c-Met. The accumulation of G(D2) in c-Met expressing cells could therefore reinforce the tumorigenicity and aggressiveness of breast cancer tumors.

Iminosugar-ferrocene conjugates as potential anticancer agents.

We prepared a series of new iminosugar-ferrocene hybrids displaying potent inhibition of fucosidase (bovine kidney) and inactivation of MDA-MB-231 breast cancer cells proliferation at low micromolar concentrations. The synthetic route brought to light an unprecedented isomerisation of a 2-ethanalylpyrrolidine.

Accumulation of unusual gangliosides G(Q3) and G(P3) in breast cancer cells expressing the G(D3) synthase.

Glycosphingolipids from the ganglio-series are usually classified in four series according to the presence of 0 to 3 sialic acid residues linked to lactosylceramide. The transfer of sialic acid is catalyzed in the Golgi apparatus by specific sialyltransferases that show high specificity toward glycolipid substrates. ST8Sia I (EC 2.4.99.8, SAT-II, SIAT 8a) is the key enzyme controlling the biosynthesis of b- and c-series gangliosides. ST8Sia I is expressed at early developmental stages whereas in adult human tissues, ST8Sia I transcripts are essentially detected in brain. ST8Sia I together with b- and c-series gangliosides are also over-expressed in neuroectoderm-derived malignant tumors such as melanoma, glioblastoma, neuroblastoma and in estrogen receptor (ER) negative breast cancer, where they play a role in cell proliferation, migration, adhesion and angiogenesis. We have stably expressed ST8Sia I in MCF-7 breast cancer cells and analyzed the glycosphingolipid composition of wild type (WT) and GD3S+ clones. As shown by mass spectrometry, MCF-7 expressed a complex pattern of neutral and sialylated glycosphingolipids from globo- and ganglio-series. WT MCF-7 cells exhibited classical monosialylated gangliosides including G(M3), G(M2), and G(M1a). In parallel, the expression of ST8Sia I in MCF-7 GD3S+ clones resulted in a dramatic change in ganglioside composition, with the expression of b- and c-series gangliosides as well as unusual tetra- and pentasialylated lactosylceramide derivatives G(Q3) (II(3)Neu5Ac(4)-Gg(2)Cer) and G(P3) (II(3)Neu5Ac(5)-Gg(2)Cer). This indicates that ST8Sia I is able to act as an oligosialyltransferase in a cellular context.

Role of GD3 Synthase ST8Sia I in Cancers.

GD3 synthase controls the biosynthesis of complex gangliosides, bearing two or more sialic acid residues. Disialylated gangliosides GD3 and GD2 are tumor-associated carbohydrate antigens (TACA) in neuro-ectoderm-derived cancers, and are directly involved in cell malignant properties, i.e., migration, invasion, stemness, and epithelial-mesenchymal transition. Since GD3 and GD2 levels are directly linked to GD3 synthase expression and activity, targeting GD3 synthase appears to be a promising strategy through which to interfere with ganglioside-associated malignant properties. We review here the current knowledge on GD3 synthase expression and regulation in cancers, and the consequences of complex ganglioside expression on cancer cell signaling and properties, highlighting the relationships between GD3 synthase expression and epithelial-mesenchymal transition and stemness. Different strategies were used to modulate GD3 synthase expression in cancer cells in vitro and in animal models, such as inhibitors or siRNA/lncRNA, which efficiently reduced cancer cell malignant properties and the proportion of GD2 positive cancer stem cells, which are associated with high metastatic properties, resistance to therapy, and cancer relapse. These data show the relevance of targeting GD3 synthase in association with conventional therapies, to decrease the number of cancer stem cells in tumors.

Molecular phylogeny and functional genomics of beta-galactoside alpha2,6-sialyltransferases that explain ubiquitous expression of st6gal1 gene in amniotes.

Sialyltransferases are key enzymes in the biosynthesis of sialoglycoconjugates that catalyze the transfer of sialic residue from its activated form to an oligosaccharidic acceptor. ß-Galactoside α2,6-sialyltransferases ST6Gal I and ST6Gal II are the two unique members of the ST6Gal family described in higher vertebrates. The availability of genome sequences enabled the identification of more distantly related invertebrates' st6gal gene sequences and allowed us to propose a scenario of their evolution. Using a phylogenomic approach, we present further evidence of an accelerated evolution of the st6gal1 genes both in their genomic regulatory sequences and in their coding sequence in reptiles, birds, and mammals known as amniotes, whereas st6gal2 genes conserve an ancestral profile of expression throughout vertebrate evolution.

Current trends in the structure-activity relationships of sialyltransferases.

Sialyltransferases (STs) represent an important group of enzymes that transfer N-acetylneuraminic acid (Neu5Ac) from cytidine monophosphate-Neu5Ac to various acceptor substrates. In higher animals, sialylated oligosaccharide structures play crucial roles in many biological processes but also in diseases, notably in microbial infection and cancer. Cell surface sialic acids have also been found in a few microorganisms, mainly pathogenic bacteria, and their presence is often associated with virulence. STs are distributed into five different families in the CAZy database (http://www.cazy.org/). On the basis of crystallographic data available for three ST families and fold recognition analysis for the two other families, STs can be grouped into two structural superfamilies that represent variations of the canonical glycosyltransferase (GT-A and GT-B) folds. These two superfamilies differ in the nature of their active site residues, notably the catalytic base (a histidine or an aspartate residue). The observed structural and functional differences strongly suggest that these two structural superfamilies have evolved independently.

Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0.

BACKGROUND: The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene. RESULTS: The cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells. CONCLUSION: Altogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these 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.