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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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Abstract

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.

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Abbreviations

BC:

Breast Cancer

BSM:

Bovine Submaxillary Mucin

CMP Neu5Ac:

cytidine-monophosphate N-acetylneuraminic acid

DMB:

1,2-diamino-4,5-methylenedioxybenzene

ER:

Estrogen Receptor

Gal:

Galactose

GalNAc:

N-acetylgalacotosamine

GD2S:

GD2 synthase

GD3S:

GD3 synthase

GT:

Glycosyltransferase

HPRT:

Hypoxanthine-guanine PhosphoRibosylTransferase

LacCer:

Lactosyl-ceramide

LC/ESI-MS:

Liquid Chromatography/ Electrospray ionization coupled to Mass Spectrometry

mAb:

monoclonal antibody

MFI:

Mean Fluorescence Intensity

Neu5Ac:

N-acetylneuraminic acid

Neu5,8Ac2 :

8-O-acetyl-N-acetylneuraminic acid

Neu5,9Ac2 :

9-O-acetyl-N-acetylneuraminic acid

OAcGD2:

O-acetyl-GD2

OAcGD3  :

O-acetyl-GD3.

TACA:

Tumor-Associated Carbohydrate Antigens

TNBC:

Triple Negative Breast Cancer

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Acknowledgements

We thank Christian Slomianny of the BICeL-Campus Lille1 (Univ. Lille, Bio Imaging Center Lille, F-59000 Lille, France) facility for access to instruments and technical advices. We are indebted to the PAGés plateform (Plateforme d’Analyses des Glycoconjugués, CNRS, UMR 8576, UGSF, Université de Lille), F-59000 Lille, France for the use of the mass spectrometer.

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Authors and Affiliations

  1. CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, University Lille, F-59000, Lille, France

    Sumeyye Cavdarli, Justine H. Dewald, Nao Yamakawa, Yann Guérardel, Philippe Delannoy & Sophie Groux-Degroote

  2. OGD2 Pharma, Institut de Recherche en Santé de l’Université de Nantes, 44007, Nantes, France

    Sumeyye Cavdarli, Mickaël Terme & Jean-Marc Le Doussal

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  1. Sumeyye Cavdarli
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  2. Justine H. Dewald
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  3. Nao Yamakawa
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Correspondence to Sophie Groux-Degroote.

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Cavdarli, S., Dewald, J.H., Yamakawa, N. et al. Identification of 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) as main O-acetylated sialic acid species of GD2 in breast cancer cells. Glycoconj J 36, 79–90 (2019). https://doi.org/10.1007/s10719-018-09856-w

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