EGF Induces Epithelial-Mesenchymal Transformation Through The PI3K-Akt/MAPK Signaling Pathways In Breast Cancer

Objectives:This study aims to investigate the signaling pathways of EGF regulating epithelial-mesenchymal transformation (EMT) in MCF-7breast cancer cells by Western blot and immunofluorescence staining.Materials and methods:The breast cancer cells MCF-7were treated with30ng/ml of EGF in vitro. Taken pictures at Oh and24h to observe cellular morphology changes by phase-contrast microscope. The expression level of mesenchymal markers, including Fibronectin, Snail, and Vimentin, and epithelial marker E-cadherin were assessed at different times by Western blot and immunofluorescence staining, respectively. Additionally, the expression levels of EMT markers were achieved by western blot in breast cancer cells which were treated by PI3DK/Akt and MAPK signaling pathways’ inhibitors, LY294002and PD98059for discussing the mechanisms and signaling pathways in EMT.Results:1. EGF changes the morphology of breast cancer cells. MCF-7breast cancer cells with and without EGF treatment were observed with phase-contrast microscope, before treatment with EGF, MCF-7cells showed tight cell-cell adhesion and significant cell polarity. However, the MCF-7cancer cells treated with30ng/ml of EGF (after24h) showed scattered and the significant morphological protrusion site. EGF treatment also induced the epithelial cells loose interaction pattern and acquired characteristics of mesenchymal cells. Our results on MCF-7cells suggest EGF especially may induce morphological changes and produce EMT.2. EGF upregulates the expression of mesenchymal markers and downregulates the expression of epithelial markers. For the mesenchymal markers, it was found that the expression of Snail protein was increased slightly after EGF treatment at2h and4h, especially at16h and24h, however, the level of Snail protein at8h was decreased. The expression of Fibronectin protein was increased after treatment with EGF at2h,4h,8h,16h, but decreased at24h and48h. Additionally, the expression of Vimentin was also increased slightly at2h and4h, significantly upregulated after treatment with EGF at8h and16h, and downregulated at24h and48h. Taken together, the expression levels of Snail, Fibronectin, and Vimentin proteins were significantly upregulated in EGF treated MCF-7cancer cells, indicating that EGF could upregulate the expression of mesenchymal markers. Additionally, immunofluorescence staining for Snail was also performed in MCF-7cancer cells. By confocal observation, it was also showed that Snail was significantly located in the nucleus after treatment with EGF. However, no any expression signal was found in untreated cells. For the epithelial marker, the decreased expression of E-cadherin was firstly observed after EGF treatment at2days, and significantly decreased after5days. For confirmation, immunofluorescence staining for E-cadherin was also performed in MCF-7cancer cells with or without EGF treatment. By confocal observation, it was also showed that E-cadherin was strongly positive and significantly expressed at the membrane of MCF-7untreated cells, however, weekly positive was found in the EGF treated cells.3. EGF stimulates activation of Smad2. We examined whether EGF can induced activation of Smad2or not. We initially sought to confirm the specificity of the p-Smad2antibody by Western blot and immunofluorescent staining. Smad2protein expression levels were no difference in MCF-7cells treated by EGF at different time. However, p-Smad2protein expression level was upregulated in the cells treated with EGF at lh,2h,4h,8h and16h, and decreased at24h and48h to the similar level of untreated cells. Additionally, immunofluorescence staining for phospho-Smad2was also performed in MCF-7cancer cells. By confocal observation, it was also showed that phospho-Smad2was significantly located in the nucleus. However, no any expression signal was found in untreated cells.4. EMT induced by EGF through the PI3K/Akt and MAPK pathways. LY294002is an effective inhibitor of PI3K/Akt signaling pathway. The MCF-7cells were treated with LY294002, and found that LY294002could effectively downregulate the expression of phospho-Akt, indicating that LY294002could block the phospho-Akt in MCF-7cells treated with EGF. PD98059is an effective inhibitor of MAPK signaling pathway. The MCF-7cells were treated with PD98059, and found that PD98059could effectively downregulate the expression of phospho-Erk, indicating that PD98059could block the phospho-Erk in MCF-7cells treated with EGF. The results showed that LY294002and PD98059were completely inhibited the expression of Fibronectin, Vimentin and Snail. Altogether, these results demonstrated that PI3K/Akt and MAPK signaling pathways are also important for EGF induced EMT in breast cancer cells.Conclusion:EGF induced EMT through the PI3K/MAPK pathway; and Smad2also have an important role in EMT.