ABSTRACT
Frizzled family receptor 7 (FZD7), a Wnt signaling receptor, is associated with the maintenance of stem cell properties and cancer progression. FZD7 has emerged as a potential therapeutic target because it is capable of transducing both canonical and noncanonical Wnt signals. In this study, we investigated the regulatory pathway downstream of FZD7 and its functional roles. We found that FZD7 expression was crucial to the maintenance of the mesenchymal phenotype, anoikis resistance, and spheroid and tumor formation in ovarian cancer (OC). We identified TWIST1 as the crucial downstream effector of the FZD7 pathway. TWIST1, a basic helix loop helix transcription factor, is known to associate with mesenchymal and cancer stem cell phenotypes. Manipulating TWIST1 expression mimicked the functional consequences observed in the FZD7 model, and overexpression of TWIST1 partially rescued the functional phenotypes abolished by FZD7 knockdown. We further proved that FZD7 regulated TWIST1 expression through epigenetic modifications of H3K4me3 and H3K27ac at the TWIST1 proximal promoter. We also identified that the FZD7-TWIST1 axis regulates the expression of BCL2, a gene that controls apoptosis. Identification of this FZD7-TWIST1-BCL2 pathway reaffirms the mechanism of anoikis resistance in OC. We subsequently showed that the FZD7-TWIST1 axis can be targeted by using a small molecule inhibitor of porcupine, an enzyme essential for secretion and functional activation of Wnts. In conclusion, our results identified that the FZD7-TWIST1 axis is important for tumorigenesis and anoikis resistance, and therapeutic inhibition results in cell death in OCs.
Subject(s)
Anoikis , Carcinogenesis/metabolism , Carcinogenesis/pathology , Frizzled Receptors/metabolism , Nuclear Proteins/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Signal Transduction , Twist-Related Protein 1/metabolism , Acyltransferases/antagonists & inhibitors , Acyltransferases/metabolism , Animals , Base Sequence , Cell Line, Tumor , Chickens , Down-Regulation/genetics , Epigenesis, Genetic , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Mesoderm/pathology , Phenotype , Proto-Oncogene Proteins c-bcl-2/metabolism , Survival Analysis , Treatment OutcomeABSTRACT
Epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, has been implicated to contribute to the molecular heterogeneity of epithelial ovarian cancer (EOC). Here we report that a transcription factor--Grainyhead-like 2 (GRHL2) maintains the epithelial phenotype. EOC tumours with lower GRHL2 levels are associated with the Mes/Mesenchymal molecular subtype and a poorer overall survival. shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype results in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, microRNA-200b/a is identified as the direct transcriptional target of GRHL2 and regulates the epithelial status of EOC through ZEB1 and E-cadherin. Our study demonstrates that loss of GRHL2 increases the levels of histone mark H3K27me3 on promoters and GRHL2-binding sites at miR-200b/a and E-cadherin genes. These findings support GRHL2 as a pivotal gatekeeper of EMT in EOC via miR-200-ZEB1.
Subject(s)
DNA-Binding Proteins/metabolism , Epithelial-Mesenchymal Transition , Histones/metabolism , MicroRNAs/metabolism , Neoplasm Proteins/metabolism , Ovarian Neoplasms/metabolism , Protein Processing, Post-Translational , RNA, Neoplasm/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Zinc Finger E-box-Binding Homeobox 1/metabolism , Cell Line, Tumor , DNA-Binding Proteins/genetics , Female , Histones/genetics , Humans , MicroRNAs/genetics , Neoplasm Proteins/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , RNA, Neoplasm/genetics , Response Elements , Transcription Factors/genetics , Zinc Finger E-box-Binding Homeobox 1/geneticsABSTRACT
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.
