Abstract
Alterations in protein glycosylation in colorectal cancer (CRC) have been extensively studied using cell lines as models. However, little is known about their O-glycome and the differences in glycan biosynthesis in different cell types. To provide a better understanding of the variation in O-glycosylation phenotypes and their association with other molecular features, an in-depth O-glycosylation analysis of 26 different CRC cell lines was performed. The released O-glycans were analysed on porous graphitized carbon nano-liquid chromatography system coupled to a mass spectrometer via electrospray ionization (PGC-nano-LC–ESI-MS/MS) allowing isomeric separation as well as in-depth structural characterization. Associations between the observed glycan phenotypes with previously reported cell line transcriptome signatures were examined by canonical correlation analysis. Striking differences are observed between the O-glycomes of 26 CRC cell lines. Unsupervized principal component analysis reveals a separation between well-differentiated colon-like and undifferentiated cell lines. Colon-like cell lines are characterized by a prevalence of I-branched and sialyl Lewis x/a epitope carrying glycans, while most undifferentiated cell lines show absence of Lewis epitope expression resulting in dominance of truncated α2,6-core sialylated glycans. Moreover, the expression of glycan signatures associates with the expression of glycosyltransferases that are involved in their biosynthesis, providing a deeper insight into the regulation of glycan biosynthesis in different cell types. This untargeted in-depth screening of cell line O-glycomes paves the way for future studies exploring the role of glycosylation in CRC development and drug response leading to discovery of novel targets for the development of anti-cancer antibodies.
Highlights
With over 18 million new cases worldwide in 2018, colorectal cancer (CRC) is the third most common cancer in the world [1]
We investigated the O-glycosylation phenotypes of 26 CRC cell lines derived from both primary tumours and metastatic sites, revealing pronounced differences between the cell lines
The well-differentiated cell lines show an overall higher expression of Lewis antigens and I-branched glycans, while the undifferentiated cell lines show a higher abundance of glycans carrying an α2,6-linked N-acetylneuraminic acids (NeuAc) to the core GalNAc
Summary
With over 18 million new cases worldwide in 2018, colorectal cancer (CRC) is the third most common cancer in the world [1]. The mesenchymal CMS4 tumours are characterized by infiltration with cancer-associated fibroblasts and upregulation of epithelial to mesenchymal transition (EMT) resulting in worse overall patient prognosis [3] Both CMS2 and CMS3 tumours show strong epithelial differentiation signatures, with characteristic metabolic pathway dysregulation in the CMS3 group [4]. The N-glycosylation of a set of CRC cell lines has been characterized, revealing association of antennary fucosylation with differentiation and caudal type homeobox 1 (CDX1) expression [12, 13] Another major class of colon glycans is mucin-type O-glycans, mainly carried by heavily glycosylated mucin proteins, which are the major components of the mucus layer in the gastrointestinal tract. Associations are found between the observed glycome phenotypes and cell line gene expression as well as their differentiation
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