Investigations into altered cellular glycosylation associated with metastatic competence in breast cancer cell lines
Metastasis is the main cause of cancer patient death owing to the fact that metastatic disease is difficult to treat. Aberrant glycosylation is characteristic of cancer and plays a role in metastatic mechanisms. HPA, a lectin from the Roman snail Helix pomatia, recognises glycans terminating in α-GalNAc, and is associated with aggressive biological behaviour, poor patient prognosis and metastasis in breast and other cancers. Three breast cancer cell lines characterised to stably synthesise an array of GalNAc-glycans were employed in the work reported here (MCF7, ZR751 and BT474). HPA lectin labelling and quantification were optimised and it revealed a more complex HPA-binding profile than previously reported. It was observed that HPA-positive cells were rounded, reminiscent of epithelial cells, and HPA-negative cells were elongated, and reminiscent of mesenchymal cells. One of the aberrant glycans that HPA reportedly binds is the initial O-glycan Tn structure which is commonly exposed in cancer. qPCR was performed to assess the gene expression levels of O-linked glycosylation initiation enzymes, GALNTs as well as C1GalT and its molecular chaperone COSMC. This analysis revealed that there is no clear association between aberrant glycosylation recognised by HPA-binding, although down-regulation of C1GalT and COSMC may result in failed normal chain extension. A method of isolating cells based on their HPA-binding profile was developed. Cells were separated and grown on post-separation for 3 days. It was observed that the glycosylation profiles of the HPA-positive cell population and the HPA-negative cell population reverted to a mixed population over time that was similar to the unseparated lectin labelling proportions. The cell morphologies also reverted. The plasticity observed was reminiscent of EMT. Microarray analysis revealed that several EMT-associated genes were differentially expressed between the HPA-positive and HPA-negative isolated cell populations. Using a Matrigel invasion assay it was observed that HPA-negative cells were significantly more invasive than HPA-positive cells. While in a static adhesion assay HPA-positive cells were significantly more adhesive to endothelial cell monolayers than HPA-negative cells. HPA-binding glycans were demonstrated to be functionally involved in HPA-positive cells adhesion to the endothelial cell monolayer as their adhesion was inhibited when HPA-binding glycans were masked. Furthermore, using conditioned medium transfer experiments it was observed that cell-cell signalling was occurring between the HPA-positive and HPA-negative cells and altering their adhesive characteristics. To conclude the work reported here demonstrates that cells can modulate their glycosylation profiles and their subsequent morphologies and that this plasticity is important in cancer metastasis.
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