Investigation of the role of CD24 gene in the progression of colorectal cancer

Abstract

Cluster of differentiation (CD) 24 is a glycosyl-phosphatidylinositol (GPI) anchor protein. CD24 is a heavily glycosylated protein, containing 16 putative O-glycosylation sites and two putative N-glycosylation sites. In colorectal cancer (CRC) and many other cancers, CD24 induces stemness, cell motility (both migration and invasion) and, through interaction with endothelium, possibly metastasis. The mechanisms by which it exerts these activities are not known. Therefore, this thesis aimed to (i) investigate the role of the putative N-glycosylation sites in mediating CD24 function, (ii) define the signalling pathways and ligands that mediate CRC cell adhesion to endothelium and metastasis, and (iii) delineate CD24 involvement in CRC angiogenesis. (i) N-glycosylation target sites on CD24 include asparagine residues at N36 and N52. Using site directed mutagenesis, each site was mutated to glutamine both individually (CD24N36Q and CD24N52Q) and in combination (CD24N[36,52]Q). The mutant clones were expressed ectopically in the CRC cell lines HCT116 and SW480 cells (CRC cell lines with low endogenous CD24 expression). The activity of the mutant CD24 was compared to known activities of wild-type CD24 (CD24WT). The expression of CD24N36Q and CD24N52Q resulted in increased cell motility compared to empty vector control (EVC) but was significantly less than that induced by CD24WT. However, the expression of CD24N[36,52]Q resulted in a near-complete loss of cell motility induction. Complementing this, analysis of downstream ix targets and F-actin staining demonstrated reduced induction of known CD24 targets and reduced cadherin-switch by the mutant vectors. (ii) CD24 is thought to act as a ligand for the endothelial molecule P-selectin to promote metastasis. To test the effect of the CD24 /P-selectin interaction on cell function, expression of CD24 was modulated by the CRC cell lines HCT116 and SW620 followed by either stimulation with recombinant P-selectin or co-culture with endothelial cells. Firstly, the interaction of P-selectin/CD24 was confirmed by co-immunoprecipitation. Adhesion assays showed that this interaction increased adhesion between epithelial and endothelial cells and that loss of either CD24 or P-selectin resulted in a reduction of adhesion. This interaction also promoted trans-endothelial migration by the epithelial cells. A positive feedback loop of P-selectin/CD24 interaction was observed, whereby P-selectin stimulation increased CD24 expression in CRCs. In addition, this interaction triggered known downstream signalling molecules associated with migration and invasion. Stimulation of the cells expressing CD24N[36,52]Q with P-selectin failed to induce cell motility and adhesion compared to CD24WT. (iii) Angiogenesis is an important part of establishing metastasis and, given CD24’s putative role in tumour metastasis, we investigated the role of CD24 in promoting angiogenesis. In order to test this role, CD24 expression was modulated in CRC cell lines (ectopically expressed in the low-expressing HCT116/ SW480 and silenced in the high-expressing SW620) and the expression of vascular endothelial growth factor (VEGF)-A (a potent x angiogenesis inducer) and angiostatin (an inhibitor of angiogenesis) were assessed by Western blot and qPCR. CD24 was shown to be an inducer of VEGF-A expression and an inhibitor of angiostatin. Transfection of cells with CD24N[36,52]Q resulted in failure to induce VEGF-A or inhibit angiostatin. The conditioned medium was collected from the above-transfected cells to study the effect of CD24-induced secreted products on endothelial cells. The conditioned medium from CD24-expressing cells significantly induced the proliferation and migration, and tube formation of endothelial cells, whereas CD24 silencing had an opposite effect. Integrin-linked kinase (ILK) has been shown to be a target o