Identification of tumour-associated gycoproteins recognised by the lectin from Helix pomatia in breast cancer

Helix pomatia agglutinin (HPA) is a carbohydrate binding protein isolated from the Roman snail. There has been considerable interest in understanding HPA binding partners in cancer, as the lectin has been shown to identify glycosylation changes in cancers arising from the epithelia, from patients with poor prognosis. Identifying the HPA binding epitopes associated with a malignant phenotype may be useful for prognostication and may also offer potential as targets for immunotherapy. Previous studies have shown that HPA recognises a multitude of proteins in colorectal cancer (CRC). This study aimed to establish whether HPA recognises the same glycoproteins in breast cancer. An in vitro model of human breast cancer cell lines was used, ranging from HPA negative, non metastatic, to HPA positive and metastatic. Four human breast cell lines were chosen to represent phenotypes ranging from ‘normal’/benign (HMT3522), primary cancer (BT474) to metastatic cancer (T47D, MCF-7). HPA binding was assessed using confocal microscopy. Membrane proteins were extracted by differential centrifugation and the proteins were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blotting. The cell surface glycoproteins recognised by HPA were characterised using 2-dimensional electrophoresis (2-DE), Western blotting and mass spectrometry. HPA binding correlated with integrin ? 6 levels, this concurred with previous findings in CRC. HPA also bound transcription factors HnRNP H1, HnRNP D-like, HnRNP A2/B1 as well as Hsp27, GFAP and ENO1. The recognition of HnRNPs, Hsp 27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Interestingly, these HPA-binding glycoproteins were either absent or showed decreased levels in the non-metastatic breast cancer cell line BT474 and in ‘normal’ HMT3522. A comprehensive analysis of the breast cells proteome showed a number of proteins with elevated level in the metastatic breast cancer cell lines T47D and MCF-7, but this is the first report to show elevated levels of elongation factor Tu, Enoyl Coenzyme A hydratase 1 peroxisomal and macropain subunits. This work was extended to analyse the gene expression for UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase (ppGalNAc T1,T2, T3 and T6) and alpha 2,6 sialyltransferases (ST6GalNAc I and II) in the breast cell lines, but no correlation between the expression levels of mRNA of these enzymes and HPA binding was found in this study. The results from the present study show that, as in CRC cell lines, integrin ? 6 was the most abundant HPA binding glycoprotein extracted from the breast cancer cells with a metastatic phenotype. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represent a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype. New approaches aimed at targeting these changes might have broad application for the treatment of breast, colorectal and possibly other epithelial cancers.