Sajid, Saira
(2018)
Understanding the biology Of CD24.
PhD thesis, University of Nottingham.
Abstract
Cancers are amongst the leading cause of morbidity and mortality today. Besides the tremendous amount of research, it still appears to be a long way till we can fully understand the pathology and find its cure. Scientists are still striving to find out the precise pathogenesis, factors leading to progression and the mechanisms of spread of cancers. The ultimate objective is to find out how these can be prevented and treated. Many molecules are a focus of attention in this regard and CD24 is amongst them. CD24 is normally present on haematopoietic cells and embryonal epithelial cells but the expression is generally lost with cellular maturity and differentiation.
Upregulation of CD24 has been documented in a large variety of cancers, besides non-malignant pathologies. In the recent literature, CD24 has been linked to significant cancer associated properies such as proliferation, metastasis and cancer stem cells. It is an interesting molecule with a very small protein core decorated with heavy glycosylation. The pattern and composition of CD24 glycosylation also varies within different tissues. This study attempted to explore the molecule a bit further regarding its structure, functions, molecular interactions and possible downstream signalling partners.
The first part of the project was to evaluate the possible effects of CD24 in mediating cellular response to DAMPs (Damage Associated Molecular Patterns) in colorectal cancer cell lines. Malignancies have increased CD24 levels and there is increased tissue damage and necrosis thereby release of DAMPs (Damage Associated Molecular Patterns) in their microenvironment. The effects of CD24 on modulation of DAMPs response were analysed by exposing cells to autologous DAMPs and performing functional assays (migration and proliferation assays).
The results of wound healing assay showed significant inhibition of colorectal cell migration by DAMPs but this was independent of CD24 status. Furthermore, transwell assays also showed significantly reduced directional motility, independent of CD24 status, in cells exposed to autologous DAMPs. The study also tested the effects of DAMPs on cellular proliferation in three colorectal cancer cell lines using Presto Blue assay. The results indicated a significant increase in cellular proliferation when exposed to DAMPs, irrespective of the CD24 status of the cell line. We proposed thereby that in colorectal cancer cells, CD24 does not appear to modulate cellular response to DAMPs unlike seen in immune cells. Nevertheless, DAMPs did show effects on autologous cell migration and proliferation. However it is acknowledged that the arguments can be strengthened by the use of purified DAMPs to demonstrate similar results and also by showing abrogation of effects after addition of anti-DAMPs antibodies to our functional assays.
Another interesting aspect of CD24 is its localisation in the cell membrane. It is attached to the cell membrane via GPI anchor and resides in lipid rafts. Interestingly, it has no cytoplasmic or transmembrane domains that are used by most signalling molecules. It is unclear how CD24 molecule mediates diverse cellular properties and molecular responses in the absence of traditional signalling domains.
The second part of this study was aimed at exploring some of the potential signalling partners and their functional relevance if any to CD24. The study explored Cten and CD24 interactions, as both molecules are proposed to be mediators of increased cellular motility. CD24 and Cten were also observed to have some common downstream signalling targets. In addition, the expression levels of both molecules also presented similar trends amid different cancer cell lines.
The above observations led us to contemplate if the two molecules have mutual signalling and functional relationship. We observed that CD24 up regulation led to increased levels of Cten protein, whereas, knock down of CD24 resulted in down regulation of Cten protein. Also the results of our functional studies showed that the knockdown of Cten in co-transfection experiments abrogated the increased cell motility by CD24. Based on these observations we proposed that CD24 appears to modulate Cten levels and this regulation has significant functional relevance as well. The Co-immunoprecipitation experiments indicated that these two molecules did not seem to have physical interaction with each other, suggesting the possibility that the regulation of Cten by CD24 may be arbitrated by intermediate molecules.
Similarly, we also investigated the molecular relationship between CD24, ILK and FAK using co-transfection technique. The results revealed enhanced cell migration through the membrane by CD24 but it was reduced after the knock down of both ILK and FAK. These findings provide further insight that not only these molecules are being regulated by CD24 as proposed by many recent studies but are also functionally relevant. The current study proposed that the increased cancer cell motility demonstrated by CD24 may well be mediated through FAK and ILK in addition to Cten. The argument needs further validation as the confirmation of successful transfection and expression levels by western blots are missing in this study. This is recognised as a limitation.
Being heavily glycosylated CD24 is considered a mucin- like molecule and many studies point towards the possibility of its functional diversity to be related to these sugars. But to date not much is known about this aspect of CD24. Hence, the third part of our study was aimed at finding the functional and signalling significance of these sugar binding sites. CD24 has both O and N-sugar binding sites. While the molecule has multiple O-glycosylation sites, there are only two potential sites for N-glycosylation. Employing the fact that for CD24 molecule to be N-glycosylated, asparagine is a must and should be present in a specific sequence. We aimed at replacing the asparagine by glutamine, hence disabling the site to be decorated by N-sugars in CD24. We designed to mutate these N-glycosylation sites using Phusion-site directed mutagenesis kit. The procedure uses mutant primers in a PCR-based methodology to create the desired mutation. Once successful the next step of the study was to use this mutant CD24 in further experiments to explore the signalling and functional significance of these sugars in CD24 molecule. However, the results of our gene sequencing experiments showed that the attempt at generating the required clones was not successful and needed retrial. This step could not be carried out due to time limitation.
In summary, it can be said the study aimed at exploring novel aspects of CD24 biology from three different perspectives with some exciting new facts and findings. Though these are undermined by a number of limitations that can be rectified as discussed in the thesis and would generate stronger and publishable contribution to the current understanding of CD24 biology.
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