Investigating the signalling and functional activity of CD24 in cancer

Otifi, Hassan (2017) Investigating the signalling and functional activity of CD24 in cancer. PhD thesis, University of Nottingham.

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Background and Aim: The cluster of differentiation 24 (CD24) is a human protein encoded by the CD24 gene which maps to chromosome 6q21. It is a small highly-glycosylated protein that is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. Various studies have proven that CD24 is overexpressed in many human tumours, and its expression level has been found to be associated with a poor prognosis. Recently, CD24 has also been identified as a stem cell marker in several types of cancer. However little is known about CD24’s biological role in cancer or the mechanism through which it acts in cancer development. Thus, the main aim of this study is to investigate the potential functional role and signalling pathways of CD24 in various cancer models, such as colorectal cancer (CRC), pancreatic cancer, liver cancer, and lung cancer.

Methods: The signalling pathway and functional role of CD24 has been studied in a total of 13 well-characterised cell lines from four cancer models i.e. cancers of colorectum, pancreas, liver and lung. To identify potential downstream targets of CD24, CD24 was forcibly expressed via transient transfection in cell lines expressing low levels of CD24 or, in contrast, it was knocked down using RNA interference (siRNA) in cell lines expressing high levels of CD24. Subsequent to CD24 manipulation, we used Western blotting and/or qPCR to investigate changes in the expression of specific proteins that have been hypothesised to be involved downstream in the CD24 signalling pathway e.g., C-terminal Tensin-like (Cten), focal adhesion kinase (FAK), integrin-linked kinase (ILK) and Src. In addition, changes in epithelial mesenchymal transition (EMT) markers were evaluated. Similarly, in order to find potential upstream regulators of CD24, the expression levels of some proteins that have been found to be associated with cancer (e.g., KRAS and EGFR) were manipulated using specific siRNAs or inhibitors/stimulators, and the changes in CD24 expression were then evaluated. The functional effect of these interventions were tested through measuring cell motility,invasion, proliferation, and stemness (by the colony-formation in agar). Physical interaction between proteins was tested by protein complex immunoprecipitation (CO-IP) and protein stability was tested using cycloheximide (CHX). Lastly, the subcellular localisation of CD24 was studied in CRC and lung cancer cell lines and, immunohistochemically, in tumour tissues of CRC (n=84) and NSCLC (n=58).

Results: Our data have shown that manipulating of the expression of CD24 in the tested cancer model cell lines resulted in a significant change in the expression level of Cten which in turn caused changes in the expression levels of ILK and FAK. Noticeable modifications to cell migration, invasion, proliferation, and colony-forming rate (all p<0.05) following CD24 manipulation have also been detected, indicating that the up-regulation of Cten, ILK and FAK expression by CD24 may reveal the mechanism via which cell functions are regulated. The up-regulation of Cten expression by CD24 was found to be due to protein stabilisation as confirmed by qPCR and CHX assy. CD24 was observed to activate AKT at Serine 473 (Ser473), rather than at the Threonine 308 (Thr308) residue, and potentially collaborate with PI3 kinase to induce the full activation of AKT. The inhibition of EGFR using a specific EGFR inhibitor, PD135053, and the stimulation of EGF using recombinant EGF in cell lines that did not harbour mutant KRAS resulted in significant modifications to CD24 expression, as well as in cell motility, suggesting that EGFR is an upstream regulator of CD24 expression. However, an inverse association between CD24 and KRAS was observed suggesting that EGFR does not signal to CD24 through KRAS. The signalling pathway would appear to be EGFR → CD24 →Cten →ILK/FAK→ AKT. These effects were seen in all of the models tested thereby confirming the role of CD24 in many cancers. In CRC and NSCLC cell lines and tissues, CD24 was found to be localised in both the cytoplasm and in the nucleus. An association between CD24 and its associated partners, including downstream targets, was observed in tumour tissues. This association was consistent with that observed following CD24 manipulation in cancer cell lines.

Conclusion: CD24 seems to be regulated by EGFR either directly or indirectly. Consequently, it regulates Cten, FAK and ILK and enhances cell motility, invasion, proliferation and stemness in various cancer model cell lines. A combination of Anti-CD24 antibody/siRNA and PI3 kinase inhibitors could be used in clinical practice as a potential therapeutic agent in the early stages of CRC. Our observations in the four cancer models were consistent, suggesting that CD24 may regulate cell functions in these models through a unique mechanism. The association between nuclear CD24 expression and cancer progression and metastasis should be explored in further studies.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Ilyas, M.
Keywords: CD antigens, Membrane proteins, Cancer development, Nuclear CD24 expression
Subjects: QS-QZ Preclinical sciences (NLM Classification) > QU Biochemistry
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 42797
Depositing User: OTIFI, HASSAN
Date Deposited: 12 Oct 2017 13:46
Last Modified: 14 Oct 2017 10:23

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