Inhibitory Effects Of Mdm2 Research Articles

1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDM2 protein.

The human MDM2 protein regulates the tumor suppressor protein p53 by restricting its transcriptional activity and by promoting p53 degradation. MDM2 is ubiquitously expressed, with its overexpression implicated in many forms of cancer. The inhibitory effects of MDM2 on p53 have been shown to involve its N-terminal p53-binding domain and its C-terminal RING domain. The presence of an intact central acidic domain of MDM2 has also been shown to regulate p53 ubiquitination, with this domain shown to directly interact with the p53 DNA-binding domain to regulate the DNA binding activity of p53. To date, little structural information has been obtained for the MDM2 acidic domain. Thus, to gain insight into the structure and function relationship of this region, we have applied solution-state NMR spectroscopy to characterize the segment of MDM2 spanning residues 215-300. These boundaries for the acidic domain were determined on the basis of consensus observed in multiple sequence alignment. Here, we report the 1H, 15N and 13C backbone and 13Cβ chemical shift assignments and steady-state {1H}-15N heteronuclear NOE enhancement factors as a function of residue for the acidic domain of MDM2. We show that this domain exhibits the hallmarks of being a disordered protein, on the basis both of assigned chemical shifts and residue-level backbone dynamics, with localized variation in secondary structure propensity inferred from chemical shift analysis.

Abstract 1434: MDM2 promotes tumor cell migration through the induction of epithelial to mesenchymal transition

Abstract Objectives: Epithelial to mesenchymal transition (EMT), which occurs in the initiation of metastasis, offers tumor cells increased invasive properties. MDM2 overexpression has been reported to predict distant metastasis, with mechanisms undefined. The present study will investigate the role of MDM2 in regulating EMT, so as to promote the discovery and development of novel targets for anti-metastasis therapy. Methods: (1) MDM2 expression in human ovarian adenocarcinoma tissues was examined by immunohistochemical staining; (2) SKOV3 cells were transfected with MDM2 plasmid, then the alteration of cell motility was detected using wound-healing and transwell assays; the protein and mRNA expression of E-cadherin and Slug/Snail were detected by Western Blot and real-time PCR respectively; (3) Smad2, Smad3 or Smad4 was silenced by siRNA, then E-cadherin protein expression affected by MDM2 overexpression was determined by Western Blot; (4) After transfection with MDM2 siRNA, TGF-β-triggered activation EMT and cell migration were observed; (5) The effect of MDM2 inhibitor Nutlin-3 on EMT and cell motility were observed by Western Blot and transwell assays. Results: MDM2 expression in 123 clinical samples was examined by immunohistochemical staining, which identified a significant correlation between MDM2 expression and clinical staging. The reduction of E-cadherin protein expression, as the indicator of the occurrence of EMT, was observed in SKOV3 cells transfected with MDM2 plasmid, which led to increased cell motility. In addition, evident upregulation of Slug and Snail expression was induced by MDM2 overexpression. Smad complexes directly activate the transcription of Slug and Snail. In our study, after silencing Smad2, Smad3 or Smad4, the inhibitory effect of MDM2 on E-cadherin expression was counteracted, indicating that Smad pathway might be critical for MDM2-promoted EMT. Then we found that TGF-β-Smad-induced EMT and cell migration could be suppressed by silencing MDM2. Further investigation suggested that the MDM2 inhibitor Nutlin-3 could prevent the downregulation of E-cadherin induced by TGF-β or MDM2 overexpression through interrupting Smad activation. Additionally, Nutlin-3 exhibited inhibitory effect on cell migration, which might be owing to its antagonism on the EMT. Conclusion: In summary, this study explored the molecular mechanisms underlying the role of MDM2 in cancer progression. MDM2 expression has been demonstrated to be closely correlated to human ovarian adenocarcinoma clinical staging, indicating the possible utilization of MDM2 in prognosis. Further study clarified MDM2 promoted tumor cell migration through the induction of EMT, which provides new theoretical evidences for the roles that MDM2 plays in tumor metastasis. Our study implicates that MDM2 may be a potential target for inhibiting metastasis and contributes to the translational research of MDM2 inhibitors as anti-metastatic agents. Citation Format: Lin Zheng, Yeping Wu, Tianyi Zhou, Hong Zhu, Qiaojun He, Bo Yang. MDM2 promotes tumor cell migration through the induction of epithelial to mesenchymal transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1434. doi:10.1158/1538-7445.AM2015-1434

Mdm2 Regulates p53 mRNA Translation through Inhibitory Interactions with Ribosomal Protein L26

Mdm2 regulates the p53 tumor suppressor by promoting its proteasome-mediated degradation. Mdm2 and p53 engage in an autoregulatory feedback loop that maintains low p53 activity in nonstressed cells. We now report that Mdm2 regulates p53 levels also by targeting ribosomal protein L26. L26 binds p53 mRNA and augments its translation. Mdm2 binds L26 and drives its polyubiquitylation and proteasomal degradation. In addition, the binding of Mdm2 to L26 attenuates the association of L26 with p53 mRNA and represses L26-mediated augmentation of p53 protein synthesis. Under nonstressed conditions, both mechanisms help maintain low cellular p53 levels by constitutively tuning down p53 translation. In response to genotoxic stress, the inhibitory effect of Mdm2 on L26 is attenuated, enabling a rapid increase in p53 synthesis. The Mdm2-L26 interaction thus represents an additional important component of the autoregulatory feedback loop that dictates cellular p53 levels and activity.

Cooperativity of p19ARF, Mdm2, and p53 in murine tumorigenesis.

The p19ARF gene product responds to oncogenic stresses by interfering with the inhibitory effects of Mdm2 on p53, thus enhancing p53 activity and its antiproliferative functions. The absence of p19ARF in the mouse leads to early tumor susceptibility, presumably in part due to decreased p53 activity. To examine the tumorigenic cooperativity of p19ARF, Mdm2, and p53 in vivo, p19ARF-deficient mice were crossed first to p53-deficient mice and then to Mdm2 transgenic mice. The progeny were monitored for tumors. Cooperativity between p19ARF and p53 deficiencies in accelerating tumor formation was observed for most genotypes except p53-/- p19ARF-/- mice. p53-/- p19ARF-/- mice had a tumor incidence similar to p53-/- mice. In this context, tumor suppression by ARF appears to be primarily p53 dependent. The majority of the p19ARF+/- tumors deleted the wildtype p19ARF allele, in agreement with the previous studies, suggesting that p19ARF is a classic 'two hit' tumor suppressor. In a p53+/- background, however, all p19ARF+/- tumors retained a wildtype ARF allele and most also retained wildtype p53. In the second cross between p19ARF-deficient and Mdm2 transgenic mice, cooperativity in tumor incidence between Mdm2 overexpression and ARF deficiency was observed, consistent with the role of p19ARF in negatively regulating Mdm2 activity. These experiments further demonstrate in vivo the inter-relationships of the p19ARF-Mdm2-p53 signaling axis in tumor suppression.

Critical role for Ser20 of human p53 in the negative regulation of p53 by Mdm2.

In response to environmental stress, the p53 phosphoprotein is stabilized and activated to inhibit cell growth. p53 stability and activity are negatively regulated by the murine double minute (Mdm2) oncoprotein in an autoregulatory feedback loop. The inhibitory effect of Mdm2 on p53 has to be tightly regulated for proper p53 activity. Phosphorylation is an important level of p53 regulation. In response to DNA damage, p53 is phosphorylated at several N-terminal serines. In this study we examined the role of Ser20, a potential phosphorylation site in human p53, in the regulation of p53 stability and function. Substitution of Ser20 by Ala (p53-Ala20) significantly increases the susceptibility of human p53 to negative regulation by Mdm2 in vivo, as measured by apoptosis and transcription activation assays. Mutation of Ser20 to Ala renders p53 less stable and more prone to Mdm2-mediated degradation. While the in vitro binding of p53 to Mdm2 is not increased by the Ala20 mutation, the same mutation results in a markedly enhanced binding in vivo. This is consistent with the conclusion that phosphorylation of Ser20 in vivo attenuates the binding of wild-type p53 to Mdm2. Peptides bearing non-phosphorylated Ser20 or Ala20 compete with p53 for Mdm2 binding, while a similar peptide with phosphorylated Ser20 does not. This implies a critical role for Ser20 in modulating the negative regulation of p53 by Mdm2, probably through phosphorylation-dependent inhibition of p53-Mdm2 interaction.