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.
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Acknowledgements
We are grateful to Drs Heide Ford and Dean Edwards for critical review of this manuscript. We thank the members of the Schedin and Gutierrez-Hartmann laboratories for their helpful comments and suggestions. We also thank Ms Rachel Burhard and Kristen Rumer for providing IHC technical assistance. DNA sequencing was provided by the Core Facility of the University of Colorado Comprehensive Cancer Center (NIH P30 CA 46934). This work was supported by grants from the NIH NCI R01 CA85944 and Cancer League of Colorado to PS, and the DOD DAMD 17-00-1-0476 to AGH. We are particularly thankful to Dr A Peter Czernilofzky (Boehringer Ingleheim Austria GmbH) for providing support to AGH during the initial phases of this work.
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Schedin, P., Eckel-Mahan, K., McDaniel, S. et al. ESX induces transformation and functional epithelial to mesenchymal transition in MCF-12A mammary epithelial cells. Oncogene 23, 1766–1779 (2004). https://doi.org/10.1038/sj.onc.1207391
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DOI: https://doi.org/10.1038/sj.onc.1207391
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