Towards improved glycan and glycoprotein biomarkers in colorectal cancer by mass spectrometry

Colorectal cancer (CRC) is a common malignancy with an estimated incidence of over one million new cases worldwide per annum. The classical diagnostic biomarker carcinoembryonic antigen (CEA) has limited sensitivity, so is not well suited for population-based screening. Therefore, there remains a need for novel biomarkers for the early detection of CRC. As protein glycosylation and glycans are underexplored as sources of potential biomarkers, this thesis uses mass spectrometry to investigate these as possible novel sources of CRC biomarkers. In the first phase of the work, N- and O-glycans from five CRC cell lines were compared with those recovered from colonic tumours (Chapter 2). To provide a bona fide comparison, the epithelial cells from the tumours were enriched using an immunoaffinity method based on EpCAM expression. Glycosylation differences were found in both N- and O-glycans. However, the differences were especially striking in the O-glycans. Amongst these differences, the well-known glycan cancer marker, sialyl-Tn was expressed in high abundance in the tumours, but it was not detected in the cell lines, except in the mucin-producing LS174T cells. Additional work using qRT-PCR implicated the regulation of glycosyltransferase and mucin expression in the formation of sialyl-Tn. Overall, the glycosylation differences found between cell line model systems and tumours, indicates that glycan markers may be differentially expressed in these two systems. This finding cautions against the use of cell lines only for biomarker discovery as glycan biomarkers may be missed. Expanding on this work, colon epithelial cells enriched from pair-matched normal and tumour tissues were investigated (Chapter 3 and 4). In Chapter 3, the N- and O-glycan profiles of epithelial cells from non-neoplastic and cancer tissues were compared. As expected, sialyl-Tn was increased in the tumours. Additionally, 13 other glycans (12 N-glycans, 1 O-glycan) were found differentially expressed in the tumours; 12 of these have not been reported previously. The changes in glycosylation also implicate changes in the corresponding biosynthetic pathways and are potential new markers for CRC. Given that glycosylation changes were associated with membrane proteins, label-free membrane proteomics was also carried out on the epithelial-enriched normal and tumour colonic tissues (Chapter 4). Over 1600 proteins were identified with 52 membrane proteins differentially expressed, which included 9 glycoproteins. Proteins previously linked with CRC were identified as well as proteins not formerly associated with CRC and these altered proteins are implicated in nucleotide binding, protein transport, immunity and cell proliferation. The N-glycosylation sites of membrane glycoproteins were also mapped, which included the confirmation of 60 potential sites and identification of 16 unannotated sites. Intriguingly, quantification of formerly glycosylated and non-glycosylated peptides from CEA suggests N-glycosylation site differences which may be used to differentiate normal and tumour tissues. In summary, this thesis examines the alterations in glycan and glycoproteins of epithelial cells in colorectal cancer using mass spectrometry. The study has developed a comprehensive workflow that has enabled new insights to be revealed about glycan, protein and glycosylation site changes in tumours. These molecular changes may provide novel leads in the ongoing search for new biomarkers of improved sensitivity and specificity in colorectal cancer.