Investigating the signalling and functional activity of CD24 in cancer

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