TIMP-2 regulates proliferation, invasion and STAT3-mediated cancer stem cell-dependent chemoresistance in ovarian cancer cells

Maree Bilandzic,Patrick Western,Jock K Findlay,George Kannourakis,Ruth M Escalona,Nuzhat Ahmed,Elif Kadife

doi:10.1186/s12885-020-07274-6

Maree Bilandzic, Patrick Western + Show 5 more

Open Access

https://doi.org/10.1186/s12885-020-07274-6

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Abstract

BackgroundThe metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines.MethodsFT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence.ResultsSixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers.ConclusionsThe above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance.

Highlights

The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression
The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance
By using Small interfering RNA (siRNA), we examined the effect of TIMP-2 knock down (T2-KD) on MT1MMP and Matrix metalloproteinase (MMP)-2 expression levels, proliferation, invasion and chemosensitivity in Fallopian tube secretory epithelial cells and two ovarian cancer cell lines

Summary

Introduction

The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. A more detailed knowledge of the biological mechanisms leading to chemoresistance is essential to achieve a better treatment outcome for persistent recurring ovarian cancer patients. Of all the TIMPs, TIMP-2 only interacts with a cell-membrane bound MMP, commonly known as MT1-MMP and can act as either an initiator or an inhibitor of MMP-2 activation. As an initiator of MMP-2 activation, the catalytic domain of MT1-MMP binds to the N-terminal region of TIMP-2. This leaves the C-terminal region of TIMP-2 free for binding to the hemopexin-like domain of pro-MMP-2 [9] This ternary MT1-MMP-TIMP2 complex facilitates assembly of the extracellular secreted pro-MMP-2 on the cell surface in close proximity to TIMP-free active MT1-MMP. In cases where TIMP-2 acts as an inhibitor of MMP-2, the C-terminal end of TIMP-2 acts as a receptor for the C-terminal region of MMP-2, binding of which prevents the interaction of TIMP-2 with MT1-MMP, thereby preventing subsequent MMP-2 activation [10]

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