Novel strategies to enforce an epithelial phenotype in mesenchymal cells.
E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definition of several known regulators of E-cadherin expression, including ZEB1, HDAC1, and MMP14. We identified three new regulators (FLASH, CASP7, and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. In addition, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a posttranscriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through posttranscriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT.
Authors' Affiliation: Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut.
Ana-Maria
AM
Dragoi
Authors' Affiliation: Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut.
Rachel
R
Swiss
Authors' Affiliation: Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut.
Beile
B
Gao
Authors' Affiliation: Department of Microbial Pathogenesis, Yale School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut herve.agaisse@yale.edu.
Hervé
H
Agaisse
D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D004847:Epithelial Cells; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D015203:Reproducibility of Results; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D059467:Transcriptome; D000071799:Zinc Finger E-box-Binding Homeobox 1
D051017:Apoptosis Regulatory Proteins; C485604:CASP8AP2 protein, human; D015820:Cadherins; D002135:Calcium-Binding Proteins; D018398:Homeodomain Proteins; D014157:Transcription Factors; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1