Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer

Ruth M Escalona; Simon Chu; Elif Kadife; Jason K Kelly; George Kannourakis; Jock K Findlay; Nuzhat Ahmed

doi:10.1186/s12935-022-02838-x

Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer

Cancer Cell Int. 2022 Dec 30;22(1):422. doi: 10.1186/s12935-022-02838-x.

Authors

Ruth M Escalona^{1

2

3}, Simon Chu⁴, Elif Kadife³, Jason K Kelly³, George Kannourakis^{3

5}, Jock K Findlay^{1

2}, Nuzhat Ahmed^{6

7

8

9}

Affiliations

¹ Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, VIC, 3052, Australia.
² Centre for Reproductive Health, Hudson Institute of Medical Research and Department of Translational Medicine, Monash University, Clayton, VIC, 3168, Australia.
³ Fiona Elsey Cancer Research Institute, Suites 23, 106-110 Lydiard Street South, Ballarat Technology Park Central, Ballarat, VIC, 3350, Australia.
⁴ Centre for Endocrinology and Metabolism, Hudson Institute of Medical Research and Department of Translational Medicine, Monash University, Clayton, VIC, 3168, Australia.
⁵ School of Science, Psychology and Sport, Federation University, Mt Helen, VIC, 3350, Australia.
⁶ Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, VIC, 3052, Australia. nuzhata@unimelb.edu.au.
⁷ Centre for Reproductive Health, Hudson Institute of Medical Research and Department of Translational Medicine, Monash University, Clayton, VIC, 3168, Australia. nuzhata@unimelb.edu.au.
⁸ Fiona Elsey Cancer Research Institute, Suites 23, 106-110 Lydiard Street South, Ballarat Technology Park Central, Ballarat, VIC, 3350, Australia. nuzhata@unimelb.edu.au.
⁹ School of Science, Psychology and Sport, Federation University, Mt Helen, VIC, 3350, Australia. nuzhata@unimelb.edu.au.

Abstract

Background: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9.

Methods: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2'-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model.

Results: Approximately 70-90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model.

Conclusions: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer.

Keywords: CRISPR/Cas-9; Invasion; MMP14; MMP2; Migration; Ovarian cancer; Proliferation; TIMP-2; siRNA.