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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

  • Sumeyye Cavdarli
  • Justine H. Dewald
  • Nao Yamakawa
  • Yann Guérardel
  • Mickaël Terme
  • Jean-Marc Le Doussal
  • Philippe Delannoy
  • Sophie Groux-Degroote
Original Article

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.

Keywords

Breast Cancer Gangliosides Antibody GD2 O-acetyl-GD2 Sialic acid 

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

Notes

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.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sumeyye Cavdarli
    • 1
    • 2
  • Justine H. Dewald
    • 1
  • Nao Yamakawa
    • 1
  • Yann Guérardel
    • 1
  • Mickaël Terme
    • 2
  • Jean-Marc Le Doussal
    • 2
  • Philippe Delannoy
    • 1
  • Sophie Groux-Degroote
    • 1
  1. 1.CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et FonctionnelleUniversity LilleLilleFrance
  2. 2.OGD2 PharmaInstitut de Recherche en Santé de l’Université de NantesNantesFrance

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