Georgiadi, Maria (2020) Using CRISPR/Cas9 to study SPOCK1 in pancreatic cancer. (MSc(R) thesis), Kingston University, .
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive tumours, with a devastating 5-year survival rate of less than 9%. This is in part the result of the development of an abundant desmoplastic stroma which surrounds, protects, and actively promotes a tumour conducive environment. The stroma is largely composed of extracellular matrix (ECM) proteins, cells such as fibroblasts and stellate cells that are activated in response to the tumour and soluble proteins. Among these is a group of non-structural proteins that play a central role in the mediating interactions between cells and the ECM. SPOCK1 is a member of the Secreted Protein Acidic and Rich in Cysteine (SPARC) family of matricellular proteins. In various tumours, several oncogenic roles have been described for SPOCK1 such as promoting invasiveness and metastasis. Clinical samples correlate SPOCK1 expression with advanced PDAC tumours and poor prognosis. However, very little is currently known on the mechanisms of action in PDAC but interactions with matrix metalloproteinases (MMP) and growth factors, and activation of the Phosphatidylinositol 3-kinase (PI3K)/Akt pathway are suspected. This research project aimed to understand the role of SPOCK1 in stromal and pancreatic cancer cell growth and adhesion. Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) gene editing technique was used to attempt to knockdown (KD) SPOCK1. However, while a preliminary T7 endonuclease 1 (T7E1) assay indicated the presence of a mutation, Clustal omega analysis of sequencing of the CRISPR/Cas9 transfected cell lines failed to show a mutation in the SPOCK1 region. Despite the lack of mutation in the SPOCK1 target region, functional assays showed effects on both cell growth and adhesion suggesting off-target binding of Cas9 to the single guide RNA (sgRNA). Several off-target gene were identified with sgRNA sequence similarity to SPOCK1. Further experiments searching for interactions between SPOCK1 protein and ECM components revealed fibronectin, fibroblast growth factor, collagen, and membrane type 1 matrix metalloproteinase as direct binding partners of SPOCK1, suggesting that the SPOCK1 protein has diverse roles in the PDAC ECM.
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