Investigation the role of CD24 in metastatic colorectal cancer

Alsulaiman, Abdullah (2018) Investigation the role of CD24 in metastatic colorectal cancer. PhD thesis, University of Nottingham.

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CD24 is a small (81 amino acids) GPI anchored protein which is involved in promoting cell motility and stemness and may be a part of the metastatic process. It is a heavily glycosylated molecule and contains numerous O-glycosylation sites together with two N-glycosylation sites. N-glycosylation is thought to be important in protein function, and therefore, the aim of this study is to (a) investigate the importance of N-glycosylation in the function of CD24, (b) identify other potentially functional sites in CD24 by deletion mapping, (c) define downstream targets of CD24, and (d) identify the extrinsic signals of which activate CD24.

(a) Through site-directed mutagenesis, we changed the glycosylated residues N32 (ACC to CAA) and Q52 (AAT to CAG) in CD24. Mutating each of these sites individually, when compared to pCCD24WT (wild-type CD24), caused a partial reduction in ability to induce cell motility and cell invasion (cell motility p=0.0001 cell invasion p=0.0001) and, unexpectedly, resulted in significantly enhanced cell proliferation (p=0.0001). Mutation of both sites resulted in a near loss of motility induction and retained cell proliferation. (b) We mapped the functional sites of CD24 by deleting seven amino acid segments of the whole of the mature peptide. Apart from the N-glycosylation sites, no other functional domains were identified which altered cell motility or proliferation. (c) Previously, in our lab it has been shown that Cten is downstream motility-inducing target of CD24. We hypothesised that CD24 may signal through the Notch pathway since Notch1 has an important role in maintaining CSCs. Results showed that forced expression of CD24 upregulates Notch1 and Cten whilst knockdown of CD24 causes loss of Notch1 and Cten expression. However, forced expression of CD24 with simultaneous knockdown of Notch1 resulted in failure to induce Cten. (d) CD24 is reported to act as a ligand of P-selectin. We found that stimulating CD24 expressing cell lines induced with P-selectin induced cell motility (p=0.0011) and caused an increased in the protein expression of downstream targets of CD24. Stimulating cell lines expressing CD24 with mutant glycosylation sites resulted in a failure to induce motility or CD24 targets.

We conclude, the removal of the N-glycosylation sites in CD24 resulted in a loss of cell migration and invasion, thereby suggesting the importance of these sites in mediating the migration and invasion functions of CD24. Unexpectedly, these mutations also appeared to stimulate cell proliferation, suggesting that wild type CD24 can functionally inhibit cell proliferation. Deletion mapping did not reveal any other functional sites on the mature CD24 suggesting that O-glycosylation is relatively affecting the glycosylation in the biology of CD24. Notch1 was to be an important downstream target of CD24 and a regulator of Cten. The binding of P-selectin with CD24 resulted in increased motility of CD24 which is also dependent on N-glycosylation.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Ilyas, Mohammad
Keywords: CD24; Metastatic process; N-glycosylation; Deletion mapping; Downstream targets; Extrinsic signals
Subjects: W Medicine and related subjects (NLM Classification) > WI Digestive system
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 55401
Depositing User: Alsulaiman, Abdullah
Date Deposited: 01 Nov 2018 13:00
Last Modified: 13 Dec 2020 04:30

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