MDM2Promotes EMT And Tumor Cell Migration By Activating Smad Pathway

Objectives:Most deaths from cancer occur as a result of metastasis. Epithelial to mesenchymal transition (EMT), which occurs at the early stage of metastasis, offers tumor cells invasive and mobility ability to detach from primary tumors. Enormous efforts have been made to explore the key molecules regulating and promoting EMT, and to develop relevant targeting therapeutic strategies against EMT and metastasis. MDM2over-expression has been reported to predict distant metastasis, with mechanisms need to be elucidated. Thus current study will investigate the role of MDM2in regulating EMT, so as to promote the discovery and development of novel targets for anti-metastasis therapy.Methods:(1) MDM2expression in human ovarian adenocarcinoma tissues has been examined and estimated by immunohistochemical staining and statistics analysis respectively;(2) SKOV3cells were transfected with MDM2plasmid to detect the alteration of cell motility using wound-healing and transwell assays;(3) Western blot and immunofluorescence staining were performed to determine the change of E-cadherin protein level affected by MDM2over-expression;(4) The impact of MDM2over-expression on E-cadherin transcriptional level and Slug/Snail protein or mRNA expression were detected by western blot and real-time PCR analysis respectively;(5) Western blot, immunofluorescence and luciferase assays were used to the activity of Smad pathway influenced by MDM2;(6) Smad2, Smad3or Smad4was silenced by small interfering RNA, then E-cadherin protein expression affected by MDM2over-expression was determined by western blot;(7) SKOV3cells were transfected with MDM2siRNA, then the occurrence of EMT and cell migration induced by TGF-β were observed;(8) Western blot, real-time PCR and luciferase assays were used to determine the TGF-β-triggered activation of Smad pathway influenced by silencing MDM2;(9) The effect of MDM2small molecular inhibitor Nutlin-3on EMT and cell motility were observed by western blot and transwell assays.Results:1. The high expression of MDM2in human ovarian adenocarcinoma tissues is relevant to clinical significanceMDM2expression in123clinical samples was examined by immunohistochemical staining. Positive expression in cell nuclear and cytoplasm was observed in most ovarian adenocarcinoma tissues, while negative expression was found in normal tissue. Manual scoring results also showed that the expression of MDM2in normal tissues can all be classified to low expression group, however,93%of ovarian adenocarcinoma samples exhibited medium or high expression. Image analysis results further demonstrated that MDM2expression in ovarian adenocarcinoma samples was much higher than that in normal ovarian tissues. In addition, χ2test identified a significant correlation between MDM2expression and clinical staging.2. MDM2induced cell migration through promoting EMTWound healing and transwell results showed that cell motility ability increased after being transfected with MDM2plasmid. The reduction of E-cadherin protein expression was observed in MDM2over-expression group through both western blot and immunofluorescence staining results, which indicated that MDM2could trigger the occurrence of EMT. Then Slug and Snail protein expression was examined and an evident augment of both protein levels could be observed in MDM2over-expression group.3. MDM2activated Smad pathwayReal-time PCR analysis was used to measure the mRNA level of Slug and Snail. MDM2could evidently up-regulate the transcriptional levels of Slug and Snail. Subsequently, the activity of Smad complexes, which directly activates the transcription of Slug and Snail, was detected using dual luciferease reporter gene assay. After normalized to Renilla luciferase activity, an apparent increase of the transcriptional activity of Smads complexes was observed in SKOV3cells transfected with MDM2plasmid. Furthermore, western blot and immunofluorescence staining results both clarified that MDM2could activate Smad pathway by inducing phosphorylation of Smad2and Smad3, while leaving the total expression of Smad2/3remain intact.4. The Smad pathway is indispensable for MDM2to induce EMTWestern blot results showed that the inhibitory effect of MDM2on E-cadherin expression level has been counteracted when Smad pathway was not able to be activated or Smads complexes failed to be formed after silencing Smad2, Smad3or Smad4in SKOV3cells by siRNA, which indicated that the Smad pathway is critical for MDM2-promoted EMT.5. TGF-β-Smad-induced EMT and cell migration could be suppressed by silencing MDM2Western blot and real-time PCR results showed that TGF-β-induced down-regulation of E-cadherin protein and mRNA levels can be prevented by silencing MDM2. And also, silencing MDM2could also negatively regulate the protein and mRNA level of Slug and Snail under TGF-β treatment. Therefore, the dual luciferease reporter gene assay was employed to determine the activity of Smads complexes. An apparent increase of the transcriptional activity of Smad complexes was observed in SKOV3cells subjected to TGF-β treatment, which was significantly prohibited by silencing MDM2. Western blot assay showed that TGF-β alone induced phosphorylation of Smad2and Smad3, denoting the activation of these two R-Smads; and the SKOV3cells transfected with MDM2siRNA reduced the levels of pSmad2and pSmad3. Transwell and wound healing assays were further performed and the results demonstrated that silencing MDM2distinctly reversed TGF-β-mediated migration. 6. Small molecular inhibitor of MDM2could suppress EMT by interfering Smad activationMDM2inhibitor Nutlin-3could prevent the down-regulation of E-cadherin protein induced by TGF-P or MDM2over-expression. Further investigation suggested that Nutlin-3interrupted the activation of R-Smads by suppressing their phosphorylation. And the increment of Slug and Snail protein in SKOV3cells were also remarkably decreased after the exposure to Nutlin-3, which indicated that the suppression of EMT by Nutlin-3was probably attributed to the observed inhibitory effects on Smad signaling pathway. Additionally, Nutlin-3exhibited inhibitory effect on cell migration from transwell assay, which might be owing to its antagonism on the EMT.Conclusion:In summary, the current study explored the molecular mechanisms and signaling pathways through which MDM2exerted its role in cancer progression. MDM2expression level has been demonstrated to be correlated to human ovarian adenocarcinoma clinical staging significantly in the present study, indicating the possible utilization of MDM2in prognosis as a biomarker. Further mechanism investigation clarified that MDM2could trigger the occurrence of EMT by activating Smad pathway, which provides new theoretical evidences for the roles that MDM2plays in tumor metastasis. The suppressing effect of MDM2inhibitor Nutlin-3on EMT and cell migration implicates that MDM2could become a potential target for inhibiting metastasis and contributes to the translational research of MDM2inhibitors as anti-metastatic agents.