Malik, Khalisha (2021) Investigating the interaction between SPARC and fibronectin in pancreatic cancer. (MSc(R) thesis), Kingston University, .
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with only 10% of patients surviving 10 years after diagnosis, and it accounts for ~6% of cancer deaths. PDAC is characterised by the formation of a dense stroma providing a significant barrier to drug delivery. However, the presence of the stroma has also been shown to inhibit metastasis. It is therefore important to specifically target tumour promoting aspects of the stroma as a potential way of treating pancreatic cancer. The matricellular protein SPARC is an important regulator of cell processes and cell-cell interactions and is upregulated in a number of tumours. It has shown to play either tumour-suppressive or tumourpromoting roles depending on tumour type. Overexpression of SPARC in PDAC is correlated to poor patient prognosis. The matrix protein fibronectin provides structural support to cells and overexpression of fibronectin promotes the dense stromal formation. Previous work by Munasinghe et al., (2020) has shown that depletion of fibronectin from serum is able to ‘switch’ the activity of SPARC from promoting cell proliferation to inducing apoptosis. In this study, we aimed to investigate the interaction between SPARC and fibronectin. Using a cell proliferation assay (BrdU), the addition of purified fibronectin ‘switches’ the activity of SPARC from promoting cancer cell proliferation to inhibiting proliferation. Further analysis of the binding of these two proteins using a solid phase binding assay (SBPA) confirmed that there is a degree of direct binding between SPARC and fibronectin, and that domains within fragment 1 of fibronectin binds to SPARC directly, but with a lesser degree. Whilst the presence of fibronectin was able to regulate the activity of SPARC, it was determined through Coomassie staining and western blotting that it is likely that other proteins are required to fully induce the switch in SPARC activity to promote cell proliferation as seen in 10% medium. Heparin is known to modulate the structure of fibronectin to expose cryptic binding sites however, we have found that the presence of heparin does not regulate the binding of SPARC to fibronectin through a BrdU assay and a solid phase binding assay. Whilst we have uncovered that SPARC and fibronectin are able to interact directly, further investigation is required to reveal the exact binding sites and form of fibronectin required for modulating the effect of SPARC.
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