Obstetrics and Gynaecology - Theses
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ItemThe role of metzincins and TIMPs in ovarian cancer( 2022)Epithelial ovarian cancer has the highest fatality-to-case ratio of all the gynaecological malignancies. In Australia, only 46% of patients survive 5 years after diagnosis of epithelial ovarian cancer. The metzincin family of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in matrix remodelling, regulation of signal-transduction pathways, angiogenesis and a range of diseases including cancers. This thesis investigated the role of metzincins and TIMPs in ovarian cancer providing insights in progression, chemoresistance and recurrence in patients. It revealed that TIMP-2, specifically, plays a role in serous epithelial ovarian cancer progression and regulates chemosensitivity of ovarian cancer cells through pathways that regulate cancer stem cell (CSC) phenotypes. In chapters 3 and 4, the expression patterns of metzincins and TIMPs were assessed in (a) FIGO stages, Silverberg grades and WHO types of primary serous ovarian carcinomas; and (b) ascites-derived tumour cells collected from chemo-naive and chemotherapy-treated recurrent patients and fluids. The expression of TIMP-2 and TIMP-3 was significantly upregulated in high-grade serous ovarian tumours compared to benign tumours. Moreover, TIMP-2 mRNA was regulated in the chemotherapy-treated, ascites-derived epithelial tumours compared to chemo-naive samples. TIMPs and MMPs were differentially expressed in fourteen ovarian cancer cell lines. Chemotherapy treatments (cisplatin or paclitaxel) modulated TIMPs and MMPs expression in association with increased expression of genes related to chemoresistance. Overall, the findings indicated that TIMP-2 was a likely candidate to be involved with ovarian tumour progression and chemoresistance. In chapter 5, TIMP-2 gene expression was transiently knocked-down by siRNA (T2-KD) in three high grade serous ovarian cancer cell lines (JHOS-2, OVCAR4 and OVCAR5) and a control cell line derived from normal oviductal epithelium (FT282). Overall, TIMP-2 downregulation significantly downregulated the expression and activation of MMP-2, but enhanced: proliferation, invasion, MMP-14 expression, induced EMT and increased sensitivity to cisplatin and paclitaxel. Chemotherapy treatments significantly enhanced the expression of TIMP-2, MMP-2, MMP-14, chemoresistant and CSC genes and induced activation of STAT3 in control cells, but remained unchanged in T2-KD cells. These results demonstrate that TIMP-2 plays a role in ovarian cancer progression and regulates chemosensitivity of ovarian cancer cells through pathways that regulate CSC phenotype. In chapter 6, TIMP-2 was edited in OVCAR5 cell line using CRISPR/Cas9 using two different guide RNAs. This resulted in two clonal cell lines, with gRNA2 (63% TIMP-2 knock-down) cells having similar characteristics as siRNA T2-KD cells. However, the functional outcome from gRNA1 (80% knock-down) was partially different. This cell line had similar MMP-2 and MMP-14 profile as gRNA2 but was resistant to paclitaxel. In addition, gRNA1 cells produced greater tumour burden than control cells in mice and a significantly shorter survival response compared to gRNA2 cells which produced small tumour burden and significantly longer survival. Collectively, these results demonstrate a complex role of TIMP-2 in ovarian cancer progression and chemoresistance. It is not only the reduction of TIMP-2 expression but the degree of reduction that influences the physiological/biological end points. Findings in this thesis have furthered understanding of TIMP-2 in ovarian tumorigenesis and laid the foundation for TIMP-2 mediated targeted therapy.