MDM2Cooperates With Transcription Factor Snaill And Twist To Induce Epithelial-mesenchymal Transition In Human Breast Cancer Cells

Objective:Overexpression of the human homologue of MDM2(murine double minute2), referred to as HDM2, occurs in more than50different types of human tumors including breast cancer, and amplification of the MDM2gene correlates with the metastatic stage. EMT (epithelial-mesenchymal transition) is one of the key factors to determine the extent of tumor invasion and metastasis, and a number of transcription factors such as Snaill and Twist are essential for EMT. Here, we aimed to identify the role of MDM2in the regulation of EMT and the relationship among MDM2, Snaill and Twist on the EMT process. Methods:We first performed Western blot to examine the protein expression of MDM2and EMT-related proteins in human breast cancer cell lines. Next, pcmv-MDM2expression plasmids were transiently transfected into MCF-7cells, and MDA-MB-231cells were transiently transfected with MDM2siRNA. Western blot and Immunofluorescence assays were used to examine the expression of MDM2and EMT-related proteins. Then, we transiently transfected pcmv-MDM2expression plasmids into MCF-7cells and MDA-MB-231cells, and evaluated the expression of Snaill and Twist by Western blot and real-time PCR analysis. In addition, we constructed a MDM2recombinant lentiviral vector and transduced MCF-7cells. Monoclonal was picked by limited dilution method, and then stable cell line with expression of exogenous MDM2was selected. Finally, we examined the expression of E-cadherin、Snaill and Twist at mRNA and protein levels. Results:1. MCF-7cells exhibited a high protein level of epithelial cell marker, whereas MDA-MB-231and MDA-MB-435cells exhibited a high protein level of mesenchymal cell markers. MDA-MB-435cells exhibited the highest protein level of MDM2, and MCF-7cells displayed higher level as compared with MDA-MB-231cells.2. Overexpression of MDM2in MCF-7cells induced morphological changes from cobblestone-like growth to spindle-shaped colonies and caused impaired expression of epithelial cell marker proteins.3. Knockdown of MDM2in MDA-MB-231cells induced morphological changes from fibroblast-like growth to cobblestone-like colonies and caused impaired expression of mesenchymal cell marker proteins.4. The transfection of MCF-7cells with pcmv-MDM2resulted in up-regulation of Snaill and Twist at mRNA and protein levels. Similar changes were observed also in MDA-MB-231cells.5. We obtained several stable cell lines named as MCF-7/MDM2.Among which MCF-7/MDM2-a was accompanied by a gain of MDM2、Snaill and Twist, and loss of E-cadherin. Moreover, the morphology of MCF-7/MDM2-a changed from cobblestone-like growth to spindle-shaped colonies. Conclusions:These data suggested that MDM2could cooperate with transcription factor Snaill and Twist to induce epithelial-mesenchymal transition in human breast cancer cells.