“Pulsed Hypoxia” Gradually Reprograms Breast Cancer Fibroblasts into Pro-Tumorigenic Cells via Mesenchymal–Epithelial Transition

Hypoxia arises in most growing solid tumors and can lead to pleotropic effects that potentially increase tumor aggressiveness and resistance to therapy through regulation of the expression of genes associated with the epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET). The main goal of the current work was to obtain and investigate the intermediate phenotype of tumor cells undergoing the hypoxia-dependent transition from fibroblast to epithelial morphology. Primary breast cancer fibroblasts BrC4f, being cancer-associated fibroblasts, were subjected to one or two rounds of “pulsed hypoxia” (PH). PH induced transformation of fibroblast-shaped cells to semi-epithelial cells. Western blot analysis, fluorescent microscopy and flow cytometry of transformed cells demonstrated the decrease in the mesenchymal markers vimentin and N-cad and an increase in the epithelial marker E-cad. These cells kept mesenchymal markers αSMA and S100A4 and high ALDH activity. Real-time PCR data of the cells after one (BrC4f_Hyp1) and two (BrC4f_Hyp2) rounds of PH showed consistent up-regulation of TWIST1 gene as an early response and ZEB1/2 and SLUG transcriptional activity as a subsequent response. Reversion of BrC4f_Hyp2 cells to normoxia conditions converted them to epithelial-like cells (BrC4e) with decreased expression of EMT genes and up-regulation of MET-related OVOL2 and c-MYC genes. Transplantation of BrC4f and BrC4f_Hyp2 cells into SCID mice showed the acceleration of tumor growth up to 61.6% for BrC4f_Hyp2 cells. To summarize, rounds of PH imitate the MET process of tumorigenesis in which cancer-associated fibroblasts pass through intermediate stages and become more aggressive epithelial-like tumor cells.