Functional relevance of a six mesenchymal gene signature in epithelial-mesenchymal transition (EMT) reversal by the triple angiokinase inhibitor, nintedanib (BIBF1120)
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Ruby Yun-Ju Huang1,2, Kuee Theng Kuay2, Tuan Zea Tan2, Mohammad Asad1, Hei Mui Tang2, Aloysius Hsien Chun Ng3, Jieru Ye2,3, Vin Yee Chung2 and Jean Paul Thiery2,4,5
1 Department of Obstetrics and Gynaecology, National University Health System, Singapore
2 Cancer Science Institute of Singapore, National University of Singapore, Singapore
3 Dean’s Office, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
4 Institute of Molecular and Cell Biology, A*STAR, Singapore
5 Department of Biochemistry, National University of Singapore, Singapore
Ruby Yun-Ju Huang, email:
Keywords: epithelial-mesenchymal transition (EMT), siRNA screen, EMT reversal, nintedanib, triple angiokinase inhibitor
Received: February 04, 2015 Accepted: May 22, 2015 Published: May 27, 2015
Epithelial-mesenchymal transition (EMT), a crucial mechanism in carcinoma progression, describes the process whereby epithelial cells lose their apico-basal polarity and junctional complexes and acquire a mesenchymal-like morphology. Several markers are considered to be authentic indicators of an epithelial or mesenchymal status; however, there is currently no comprehensive or systematic method with which to determine their functional relevance. Previously, we identified a 33-gene EMT signature comprising 25 epithelial and 6 mesenchymal genes that best describe this concept of the EMT spectrum. Here, we designed small-scale siRNA screens targeting these six mesenchymal signature genes (CD99L2, EMP3, ITGA5, SYDE1, VIM, ZEB1) to explore their functional relevance and their roles during EMT reversal by nintedanib (BIBF1120) in a mesenchymal-like SKOV3 ovarian cancer cell line. We found that neither cell proliferation nor cytotoxicity was affected by silencing any of these genes. SKOV3 cells expressing siRNA against mesenchymal genes (ZEB1, EMP3, CD99L2, ITGA5, and SYDE1) showed enhanced colony compaction (reduced inter-nuclear distance). Inductions of E-cadherin expression were only observed in SYDE1- and ZEB1-silenced SKOV3 cells. In addition, only SYDE1-silenced SKOV3 cells showed increased anoikis. Finally, we identified that SYDE1 and ZEB1 were down-regulated in nintedanib-treated SKOV3 cells and SYDE1- and ZEB1-silenced SKOV3 cells showed enhanced nintedanib-induced up-regulation of E-cadherin. Nintedanib-treated SKOV3 cells also showed colony compaction and decreases in EMT scores both in vitro and in vivo. We conclude that SYDE1 and ZEB1 are functionally relevant in EMT reversal. This study thus provides a proof-of-concept for the use of in vitro siRNA screening to explore the EMT-related functions of selected genes and their potential relevance in the discovery of EMT reversing drugs.
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