Eighty per cent of ovarian cancer patients die within the first five years of diagnosis due to recurrence associated with chemoresistance. Metzincin family of metalloproteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) are key modulators of biological processes in cancer progression and chemoresistance. We hypothesize that alterations in the expression of TIMP-2 is associated with ovarian tumour progression and chemoresistance.
The aims of this study was to transiently knock down (KD) the level of TIMP-2 expression in established ovarian cancer cell lines, and to (a) characterize these cell lines in relation to MMP-14/MMP-2 expression and activation; and to (b) determine the effects of chemotherapy treatment by in vitro assays.
Three ovarian cancer cell lines (FT282, JHOS-2 and OVCAR4) were transiently transfected using TIMP-2 RNAi. 80% knock down of TIMP-2 gene across all three cell lines was confirmed using single or pool siRNAs against TIMP-2 by real time PCR and immunofluorescence. MTT assays revealed an enhanced proliferation in KD cell lines compared to controls. TIMP-2 KD cells exhibited downregulation of MMP2 protein and mRNA expression. Zymography revealed inactivation of active MMP-2 levels in the KD cells. In contrast, MMP-14 protein and mRNA levels were upregulated in KD cells. All three KD cell lines exhibited increased sensitivity to cisplatin and paclitaxel compared to control.
To understand the chemosensitivity phenomenon, the mock transfected and TIMP-2 KD cells were further analysed for the expression of TIMP-2 MMP-2, MMP-14, drug resistant and cancer stem cell marker genes. These genes were up regulated in mock and control cells in response to chemotherapy, but remained unchanged in TIMP-2 KD cells. Our results demonstrate that TIMP-2 plays a role in ovarian cancer progression and regulates chemosesitivity of ovarian cancer cells through pathways that regulate cancer stem cell phenotype.