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CCAAT/enhancer binding protein beta (C/EBPβ)-2 transforms normal mammary epithelial cells and induces epithelial to mesenchymal transition in culture

Abstract

The transcription factor CCAAT/enhancer binding protein (C/EBP)β is critical for normal growth and differentiation of the mammary gland. The intronless C/EBPβ gene encodes a single mRNA that produces three protein isoforms, C/EBPβ-1, -2, and -3, which share a common basic-leucine zipper domain at their C-terminus, but are distinguished at their N-termini by the in-frame methionine codon used to initiate translation. Although C/EBPβ-1 and -2 are both transactivators, they likely perform distinct functions in mammary epithelial cells. C/EBPβ-1 is the only isoform detected in normal human mammary tissue. In breast cancer cell lines, C/EBPβ-1 is absent, and the C/EBPβ-2 transactivator is expressed. Moreover, our data suggest that C/EBPβ-2 is upregulated in human primary breast tumors. To assess C/EBPβ-2′s ability to participate in the transformation process, we generated recombinant retrovirus selectively encoding epitope-tagged C/EBPβ-2. Strikingly, 10 days after infecting a normal human mammary epithelial cell line (MCF10A) with C/EBPβ-2 virus, transformed subcultures were readily generated. Specifically, C/EBPβ-2-overexpressing MCF10A cells form foci, gain anchorage independence, express markers associated with having undergone an epithelial-to-mesenchymal transition, and acquire an invasive phenotype. These studies, and our previous observations, provide supportive evidence that deregulated expression of C/EBPβ-2 contributes to malignant conversion of the human breast.

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Acknowledgements

The authors are indebted to Katie Tipton for technical assistance. We thank Erin Eaton and Mary Hanlon for their insightful comments and careful reading of the manuscript. We are grateful for the generous gifts of the LZRS-BMN-lacZ retroviral vector and Phoenix cells from Gary Nolan (Stanford University), and vimentin antibody from Al Reynolds (Vanderbilt). We also thank William Russell, and Carlos Arteaga and his lab members for generously sharing their equipment to generate the phase contrast microscopic images. The fluorescent images from confocal and epifluorescent microscopy were acquired through the use of the VUMC Cell Imaging Core Resource (supported by NIH Grants CA68485 and DK20593) with technical assistance provided by Jonathan Sheehan and Sam Wells. Statistical analysis was carried out by Yu Shyr through the use of the VICC Biostatistics Shared Resource (supported by NCI Grant P30 CA68485).

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Correspondence to L Sealy.

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Bundy, L., Sealy, L. CCAAT/enhancer binding protein beta (C/EBPβ)-2 transforms normal mammary epithelial cells and induces epithelial to mesenchymal transition in culture. Oncogene 22, 869–883 (2003). https://doi.org/10.1038/sj.onc.1206216

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Keywords

  • C/EBP transcription factor
  • anchorage independence
  • EMT
  • invasion
  • mammary gland
  • retroviral vector

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