Epithelial to mesenchymal transition (EMT) is considered to be important for cancer invasion and metastasis. Tumour hypoxia, in addition to Transforming Growth Factor-β (TGF-β) and Notch, amongst others, have been suggested to be involved in EMT. We therefore investigated if hypoxia, TGF-β1 and the Notch ligand Jagged-1 alone induced morphological changes with corresponding EMT signatures in different epithelial breast cancer cell lines in vitro. Furthermore, we also studied whether or not TGF-β1, or Jagged-1 in combination with hypoxia added any effect on EMT.Methods:
The cells were exposed to normoxia or hypoxia alone or in combination with TGF-β1 or Jagged-1. Morphological responses to treatment were investigated by light microscopy, and changes in markers for EMT and hypoxia were evaluated by western blot analysis and immunofluorescence studies.Results:
One of the four cell lines (MCF7) became elongated and highly multipolar, indicative of EMT, following hypoxia, TGF-β1 and Jagged-1 treatment per se with the most distinct morphological shift seen with Jagged-1 treatment in combination with hypoxia. Also, when regarding hypoxia, MCF7 cells showed the greatest change in EMT-markers of the four cell lines tested, but these changes were not consistent with a typical EMT pattern. The morphology of BT474 cells was not altered following Jagged-1 treatment, however, Jagged-1 increased E-cadherin levels. Morphology was changed following TGF-β1 treatment of BT474 cells, but it did not affect E-cadherin levels. Neither Jagged-1 nor TGF-β1 altered the levels of Vimentin in the BT474 cell line. The E-cadherin responses to hypoxia varied with end-point in both MCF7 and BT474 cells, and in most cases were not consistent with EMT.Conclusion:
Our results using four different breast cancer cell lines in vitro do not provide evidence that EMT is induced by hypoxia alone or in combination with TGF-β1 or the Notch ligand Jagged-1. The inconsistency in morphological appearance and EMT-markers, as well as the time dependent variation in E-cadherin responses could not support EMT. Importantly, there was not one single common response pattern to the stimuli used, suggesting that cell lines with different hormone statuses display individual traits that respond differently to the stimuli applied. Thus, based on the present results, common statements that single factors by themselves can induce EMT seem questionable.