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Functional relevance of a six mesenchymal gene signature in epithelial-mesenchymal transition (EMT) reversal by the triple angiokinase inhibitor, nintedanib (BIBF1120).

ABSTRACT: 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.

SUBMITTER: Huang RY

PROVIDER: S-EPMC4673149 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Functional relevance of a six mesenchymal gene signature in epithelial-mesenchymal transition (EMT) reversal by the triple angiokinase inhibitor, nintedanib (BIBF1120).

Huang Ruby Yun-Ju RY Kuay Kuee Theng KT Tan Tuan Zea TZ Asad Mohammad M Tang Hei Mui HM Ng Aloysius Hsien Chun AH Ye Jieru J Chung Vin Yee VY Thiery Jean Paul JP

Oncotarget 20150901 26

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 ...[more]

PMID: 26061747

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Project description:Parathyroid hormone-related protein (PTHrP) plays a major role in skeletal metastasis but its action mechanism has not been fully defined. We previously demonstrated the crucial importance of PTHrP in promoting mammary tumor initiation, growth, and metastasis in a mouse model with a mammary epithelium-targeted Pthlh gene ablation. We demonstrate here a novel mechanism for bone invasion involving PTHrP induction of epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) regulation. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated Pthlh gene ablation was used to study EMT markers, phenotype, and invasiveness in two triple-negative breast cancer (TNBC) cell types (established MDA-MB-231 and patient-derived PT-TNBC cells). In vitro, Pthlh ablation in TNBC cells reduced EMT markers, mammosphere-forming ability, and CD44high/CD24low cells ratio. In vivo, cells were injected intratibially into athymic nude mice, and therapeutic treatment with our anti-PTHrP blocking antibody was started 2 weeks after skeletal tumors were established. In vivo, compared to control, lytic bone lesion from Pthlh -ablated cells decreased significantly over 2 weeks by 27% for MDA-MB-231 and by 75% for PT-TNBC-injected mice (p < 0.001). Micro-CT (μCT) analyses also showed that antibody therapy reduced bone lytic volume loss by 52% and 48% for non-ablated MDA-MB-231 and PT-TNBC, respectively (p < 0.05). Antibody therapy reduced skeletal tumor burden by 45% and 87% for non-ablated MDA-MB-231 and PT-TNBC, respectively (p < 0.002) and caused a significant decrease of CSC/EMT markers ALDH1, vimentin, and Slug, and an increase in E-cadherin in bone lesions. We conclude that PTHrP is a targetable EMT molecular driver and suggest that its pharmacological blockade can provide a potential therapeutic approach against established TNBC-derived skeletal lesions. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Dataset Information

Functional relevance of a six mesenchymal gene signature in epithelial-mesenchymal transition (EMT) reversal by the triple angiokinase inhibitor, nintedanib (BIBF1120).

Publications

Functional relevance of a six mesenchymal gene signature in epithelial-mesenchymal transition (EMT) reversal by the triple angiokinase inhibitor, nintedanib (BIBF1120).

Similar Datasets

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