title: ESX induces transformation and functional epithelial to mesenchymal transition in MCF-12A mammary epithelial cells.
abstract: ESX is an epithelial-restricted member of a large family of transcription factors known as the Ets family. ESX expression has been shown to be correlated with Her2/neu proto-oncogene amplification in highly aggressive breast cancers and induced by Her2/neu in breast cell lines, but its role in tumorigenesis is unknown. Previously, we have shown that ESX enhances breast cell survival in colony-formation assays. In order to determine whether ESX can act as a transforming gene, we stably transfected MCF-12A human mammary epithelial cells with the ESX expression vector, pCGN2-HA-ESX. The MCF-12A cell line is immortalized, but nontransformed, and importantly, these cells fail to express endogenous ESX protein. We used pCGN2-HA-Ets-2 and pSVRas expression vectors as positive controls for transformation. Like HA-Ets-2 and V12-Ras, stable expression of ESX induced EGF-independent proliferation, serum-independent MAPK phosphorylation and growth in soft agar. Additionally, stable ESX expression conferred increased cell adhesion, motility and invasion in two-dimensional and transwell filter assays, and an epithelial to mesenchymal morphological transition. In three-dimensional cultures, parental and vector control (pCGN2) cells formed highly organized duct-like structures with evidence of cell polarity, ECM adhesion-dependent proliferation and cell survival, and lack of cellular invasion into surrounding matrix. Remarkably, the ESX stable cells formed solid, disorganized structures, with lack of cell polarity, loss of adhesion junctions and cytokeratin staining and loss of dependence on ECM adhesion for cell proliferation and survival. In addition, ESX cells invaded the surrounding matrix, indicative of a transformed and metastatic phenotype. Taken together, these data show that ESX expression alone confers a transformed and in vitro metastatic phenotype to otherwise normal MCF-12A cells.
authors.affiliation: Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA. schedinp@amc.org
authors.forename: Pepper J
authors.initials: PJ
authors.lastname: Schedin
authors.forename: Kristin L
authors.initials: KL
authors.lastname: Eckel-Mahan
authors.forename: Shauntae M
authors.initials: SM
authors.lastname: McDaniel
authors.forename: Jason D
authors.initials: JD
authors.lastname: Prescott
authors.forename: Kelley S
authors.initials: KS
authors.lastname: Brodsky
authors.forename: John J
authors.initials: JJ
authors.lastname: Tentler
authors.forename: Arthur
authors.initials: A
authors.lastname: Gutierrez-Hartmann
mesh_terms: D001940:Breast / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002465:Cell Movement; D002471:Cell Transformation, Neoplastic; D003260:Contact Inhibition; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D008648:Mesoderm / Q000166:cytology / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009361:Neoplasm Invasiveness / Q000473:pathology; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D000090063:Proto-Oncogene Mas; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C105529:ELF3 protein, human; C107465:ERF protein, human; C494069:ETS2 protein, human; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D020928:Mitogen-Activated Protein Kinases; D015689:Oncogene Protein p21(ras)