| dc.description.abstract | This thesis encompasses an investigation into the function of plasminogen activator inhibitor 1 (PAI-1) - a serine protease inhibitor integral to coagulation and fibrinolysis that has been identified as both a key factor in and a potential regulator of the metastatic process in high-grade serous ovarian cancer (HGSOC), and also investigates the potential role of platelets in the tumour microenvironment and in haematogenous metastasis. Herein, evidence is provided in support of the hypothesis that platelet-cancer cell interactions aid survival of CTCs in the blood, and that PAI-1 is a key driver of the metastatic process in epithelial ovarian cancer (EOC) with potential as an independent prognostic indicator.
For the purpose of this study, SK-OV-3 cells were used as a model of HGSOC. A panel of assays in which PAI-1 was transiently silenced via siRNA was employed to examine the downstream effects of silencing PAI-1. These assays included real-time polymerase chain reaction (RT-qPCR), migration, invasion, wound-healing, cell-cycle analysis by flow cytometry, differential gene expression via ribonucleic acid (RNA) sequencing, and protein evaluation in both the cell line and patient cohorts by enzyme-linked immunosorbent assay (ELISA). Also interrogated were the behaviours and gene signatures of ovarian cancer cells that had been treated with consented healthy donor platelets isolated from whole blood, both in the context of otherwise untreated SK-OV-3 cells and in cells that had been treated with PAI-1 siRNA.
Results of all assays demonstrated that PAI-1 is a significant factor in the migration, invasion, wound-healing, and proliferation of ovarian cancer cells in vitro, and also demonstrated that the presence of platelets increases all of these processes. This suggested that platelet-cancer cell interactions increase PAI-1 in ovarian cancer cells, and also suggested that PAI-1 plays a central role in ovarian cancer metastasis. SERPIN E1, which encodes PAI-1 protein, was found to be significantly downregulated when PAI-1 was silenced via siRNA and significantly upregulated when platelets were co-incubated with otherwise untreated cells. Silencing PAI-1 resulted in the significant downregulation of many biological processes, including regulation of wound healing, regulation of cell-matrix adhesion, regulation of cell migration, and positive regulation of angiogenesis. Adding platelets to SK-OV-3 cells resulted in the significant upregulation of several molecular pathways, including cell migration, angiogenesis, regulation of response to wounding, and extracellular matrix organization.
In light of these results, plasma PAI-1 was evaluated in patient cohorts in order to assess any association between plasma PAI-1 and metastatic disease and also to gain insight into the potential value of PAI-1 as a prognostic indicator. These cohorts included treatment-naïve patients, those who had undergone neoadjuvant chemotherapy, patients with recurrent disease, and a fourth cohort of patients with benign histology. A pilot study in a fifth, smaller cohort of breast cancer patients sought to correlate patient plasma PAI-1 with circulating tumour cells (CTCs). Plasma PAI-1 was found to be significantly higher in patients with advanced disease compared to that of patients with early-stage disease, implying a potential role for PAI-1 as a marker of advanced disease. It was also found to be significantly reduced in patients who had undergone neoadjuvant chemotherapy compared to treatment-naïve patients, suggesting a role for PAI-1 as a marker of treatment response. Plasma PAI-1 did not correlate with patient platelet counts. A positive Pearson correlation between plasma PAI-1 and neutrophils was observed. A Kaplan-Meier overall survival (OS) analysis of PAI-1 in late-stage patients in the treatment naïve cohort trended towards significance, suggesting a poorer prognosis for patients with higher plasma PAI-1. Preliminary results of a CTC study in a 5th, smaller cohort demonstrated a significant positive correlation between plasma PAI-1 and CTC counts in patients with breast cancer. This study is currently being expanded to include patients with HGSOC. These results provide evidence for a regulatory role for PAI-1 in the processes central to ovarian cancer metastasis, and further suggest that PAI-1 may have potential value as a prognostic indicator and in the management of HGSOC. | en |
