Human TNBC may have characteristics of epithelial-mesenchymal transition (EMT).4, 5
EMT is an essential developmental process by which cells of epithelial origin lose epithelial characteristics and acquire a mesenchymal phenotype with fibroblast-like morphology, cytoskeleton reorganization, increased motility, invasiveness, and metastatic capability.7, 8
EMT is characterized by loss of epithelial cell junction proteins (e.g., E-cadherin and cytokeratins) and gain of mesenchymal markers (e.g., vimentin and fibronectin).7, 8
It has been proposed that EMT-like processes allow tumor cells to disassemble and migrate to tissue or organ sites distant from the primary tumor (metastasis).7, 8
It has become increasingly clear that EMT may play a major role in invasion and metastasis of TNBC.3-5
We have previously shown that epidermal growth factor stimulates EGFR-expressing TNBC cells such as SUM149, MDA-MB-468, and BT-20 and induces upregulation of phosphorylation of ERK and/or Akt. Further, when these cells are treated with the EGFR tyrosine kinase inhibitor (TKI) erlotinib, ERK and/or Akt phosphorylation can be downregulated. These data suggest that the EGFR pathway is intact in these cells.9, 10
To further elucidate EGFR pathway activity in TNBC, we examined whether TNBC cells (SUM149) exhibited epithelial or mesenchymal phenotypes. SUM149 cells were plated using a three-dimensional (3D) culture system. The 3D culture was performed with 2% Matrigel in F12 medium containing 5% FBS. Matrigel is a gelatinous protein mixture secreted by mouse tumor cells, which resembles the complex extracellular environment found in many tissues. This 3D culture system can be used to assess cell invasiveness and motility, which reflect part of the epithelial-to-mesenchymal morphological process due to the rapid formation of projections and filopodia. Such projections or filopodia also may strongly correlate with metastatic potential.11-14
In a two-dimensional (2D) culture system, using standard culture plates with F12 medium containing 5% FBS, SUM149 cells showed an epithelial phenotype characterized by the localization of E-cadherin and β-catenin at the sites of cell-cell contact (data not shown). However, in a 3D-culture system, SUM149 cells rapidly formed projections or filopodia and changed to a mesenchymal-like phenotype (Fig. A) within 24 hours.
Fig 1 EGFR inhibitor induced a change from mesenchymal to epithelial phenotype in TNBC cells. A,SUM149 TNBC cells were grown (3D culture) in the presence of 0 or 0.1 μM erlotinib, an EGFR tyrosine kinase inhibitor. Erlotinib (0.1 μM) inhibited (more ...)
In 3D culture, SUM149 cells with projections exhibited reduced E-cadherin expression and increased vimentin expression compared with SUM149 cells in 2D culture. Also, β-catenin accumulated at the cytoplasm in 3D culture (Fig. B).
However, when these cells were treated with the EGFR-TKI erlotinib, the mesenchymal cell phenotype changed back to epithelial-like in the 3D system, E-cadherin was upregulated, vimentin was downregulated, and β-catenin localized to the cell membrane (Fig. B). Interestingly, the erlotinib concentration that inhibited the mesenchymal phenotype (0.1 μM) did not have a cytotoxic effect and was one log lower than the concentration that inhibited proliferation (1 μM).15