Investigations into the Functional Significance and Plasticity of O-linked Glycosylation

Cell surface glycans have important roles in a myriad of biological functions including cell adhesion, signalling and identity. Mucin-type O-linked glycosylation is initiated by the attachment of an N-acetylgalactosamine (GalNAc) to a serine or threonine. This forms an initial structure called Thomsen-nouvelle (Tn) antigen. In healthy tissue, the Tn antigen is cryptic and further elaborated into linear or branched glycans. Failure in O-GalNAc glycan extension results in truncation of O-GalNAc glycans and exposure of O-GalNAc glycans, such as the Tn antigen, which can cause dysregulation of biological functions. For example, O-GalNAc glycans have been associated with poor long-term breast cancer prognosis. Model breast epithelial cell lines were investigated for their cell surface levels of O-GalNAc glycans. Model epithelial breast cancer cell lines demonstrated significantly higher levels of O-GalNAc glycans compared to the model normal breast epithelial cell line. Levels of O-GalNAc glycans present on the cell surface impacted the observable morphology of a cell. Cell shape changes are key for the epithelial-mesenchymal transition (EMT), which is implicated in wound healing and cancer metastasis. Further investigations into the relationship of EMT with a cell’s glycosylation status were conducted. Cells with high levels of O-GalNAc glycans display a more rounded, epithelial-like morphology, have high levels of E-cadherin (epithelial marker), high adhesive potential to endothelial monolayer in a static adhesion assay, and slower migratory capabilities in wound healing assays. Cells with low levels of O-GalNAc glycans demonstrated an elongated, mesenchymal-like morphology, lower levels of E-cadherin, high levels of vimentin (mesenchymal marker), high migratory capability and less adhesive to endothelial monolayers. Transforming growth factor-β1 (TGF-β1) treatment, a known inducer of EMT, of epithelial breast cancer cell lines resulted in the significant reduction of O-GalNAc glycans and E-cadherin. It also resulted in cell morphology changes, to an elongated, mesenchymal-like phenotype. RNA-sequencing revealed EMT-related genes, relating to mesenchymal markers and EMT signalling pathways, were differentially expressed in O-GalNAc glycan-negative and TGF-β1 treated cell populations compared to their O-GalNAc glycan-positive counterpart. Differentially expressed gene enrichment analysis also showed enrichment for signalling pathways associated with cancer stem cells and altered proteoglycans and ECM interactions. All these link to the functional migratory differences exhibited in O-GalNAc glycan-negative cells. The results in this thesis demonstrate the functional significance of a cell's O-GalNAc glycosylation profile in EMT and cell motility, which is of critical importance in cancer metastasis. The exposure or elaboration of O-linked glycan chains also appears to be highly plastic and dynamic allowing cancer cells to access phenotypes optimal for each stage of the metastatic cascade.

Grantor dc:publisher

Oxford Brookes University

Author dc:creator

• Cull, Joanna

Contributors dc:contributor

• Pink, Ryan
• Samuel, Priya
• Brooks, Susan

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/XZ9F-QV40

OAI identifier oai:identifier

tle:2c45fa8d-a33b-4d5c-90ed-6818d3cc18ef:d6bd9758-527a-46cd-bfe2-c433766e8fca:1