MDM2 Upregulates Snail Expression And Induces Epithelial-To-Mesenchymal Transition In Breast Cancer Cells In Vitro And In Vivo

Background:Globally, breast cancer is the most common malignancy and the second leading cause of cancer deaths in female. A well-recognized mechanism for initiating tumor cell invasive and metastatic behavior is epithelial-mesenchymal transition (EMT), in which polarized epithelial breast cancer cells acquire a motile mesenchymal phenotype. The MDM2 gene amplification occurs in diverse human malignancies, including soft tissue sarcomas and cancers of the brain, breast, ovary, cervix, lung, colon, prostate and kidney. In our previous study, we demonstrated that MDM2 promotes invasion and metastasis of breast cancer by upregulating MMP9 expression. Whether MDM2 influences other process of breast cancer metastasis requires further exploration.Objective:To explore the role of MDM2 in epithelial-to-mesenchymal transition of human breast cancer cells and its molecular mechanism.Methods:We first detected the expression of MDM2 in three breast cancer cells and one normal breast cell by quantitative RT-PCR and western blotting. Then, MCF-7 cells were infected with pRDI292-CMV or pRDI292-CMV-MDM2 lentiviruses, and the sub-clonal cells were established by puromycin selection. The morphology of cell lines were observed by light microscopy and the expression of E-cadherin, N-cadherin, vimentin and Snail were detected by quantitative RT-PCR and western blotting. Next, we knocked down MDM2 expression in MDA-MB-231 cells by RNA interference. The morphology of cell lines were observed by light microscopy and the expression of E-cadherin, N-cadherin, vimentin and Snail were detected by quantitative RT-PCR and western blotting. Then we knocked down MDM2 expression in MCF-7-MDM2-a cells by RNA interference. The morphology of cell lines were observed by light microscopy and the expression of E-cadherin, N-cadherin, vimentin and Snail were detected by quantitative RT-PCR and western blotting. Next, we knocked down Snail in MCF-7 cells and then transfected MDM2 plasmid, the EMT markers were detected by quantitative RT-PCR and western blotting. Then we knocked down NF-kappaB/P65 subunit in MCF-7 cells and then transfected MDM2 plasmid, the transcription factor Snail was detected by quantitative RT-PCR and western blotting. Next, we xenografted the MCF-7-MDM2-a and MCF-7-pCMV cells into nude mice. Nude mice were killed after six weeks, the tumor size and weight were measured, the expressions of E-cadherin, N-cadherin, vimentin and Snail were detected by quantitative RT-PCR, western blotting and immunohistochemical analysis. Finally, we stained for the expression of five genes (MDM2, E-cadherin, N-cadherin, Vimentin and Snail) and explore there correlation by Spearman correlation analysis.Results:The stable overexpression of MDM2 in MCF-7 cells (designated as MCF-7-MDM2-a,MCF-7-MDM2-d and the control (designated as MCF-7-pCMV) were established. The results showed that up-regulation of MDM2 in MCF-7 cells altered the cell morphology to a mesenchymal phenotype. Knockdown of MDM2 in MDA-MB-231 cells altered the cell morphology to the epithelial phenotype. In addition, overexpression of MDM2 increased the expression of N-cadherin and Vimentin and decreased the expression of E-cadherin, at both the mRNA and protein levels, in vitro and in vivo. Conversely, down-regulation of MDM2 decreased the expression of N-cadherin and Vimentin, and increased the expression of E-cadherin in vitro. Furthermore, MDM2 up-regulated both the mRNA and protein expression of Snail in vitro and in vivo.Knockdown of Snail almost abolished MDM2 induced EMT in vitro and knockdown of NF-kappaB/P65 subunit almost abolished MDM2 induced upregulation of Snail in MCF-7 cells. Finally, we found that MDM2 expression correlated with EMT markers and Snail:Snail expression was inversely associated with E-cadherin in human breast cancer samples.Conclusions:MDM2 can upregulate Snail by NF-kappa/B P65, enhanced Snail can induce EMT in breast cancer cells in vitro and in vivo. Thus, MDM2 may be a potential target for therapy against human metastatic breast cancer.