Abstract
Abstract Background & Aims: The Mouse Double Minute homolog 4 (MDM4) is one of the main negative p53 regulators in mammalian cells and mutational inactivation of p53 is a rare event in Western hepatocellular carcinomas (HCCs). 1q gains promote the upregulation of MDM4 in HCC, leading to a negative regulation of p53 activity. However, additional mechanisms might be involved that explain the increased MDM4 activity observed in HCCs with balanced MDM4 gene locus. Here we aimed at the identification of post-transcriptional mechanisms involved in the upregulation of MDM4 in HCC. Methods: To investigate a potential role of PI3K-AKT-mTOR signaling on MDM4 activity, HCC cell lines were treated with small molecule inhibitors and siRNAs and expression changes as well as functional analyses were recorded. Expression changes of central pathway components were investigated in human liver samples (normal liver, peritumorous liver tissue, and HCC). An AKT transgenic mouse model was used to show the involvement of AKT pathway in the regulation of MDM4 in vivo. Results: The inhibition of both PI3K-AKT and mTOR signaling pathways resulted in reduced MDM4 protein levels in HCC cell lines, which was associated with the transcriptional activation of p53-target genes. Biochemical assays revealed that both AKT-mediated phosphorylation and ubiquitin-specific protease 2a (USP2a)-mediated deubiquitination protected MDM4 from proteasomal in human HCC cell lines. In addition, AKT transgenic mice showed increased MDM4 protein levels indicating that AKT signaling is involved in the stabilization of MDM4 protein in vivo. Furthermore, the Eukaryotic translational Elongation Factor 1 alpha 2 (EEF1A2), which is frequently upregulated in human HCC, sustained the PI3K-AKT-mTOR cascade both in vitro and in vivo. In human HCCs, a strong positive correlation between the overexpression of EEF1A2, pAKT, USP2a, and MDM4 was observed, which was associated with shorter survival of HCC patients. Conclusions: Our data demonstrate that the EEF1A2/PI3K/AKT/mTOR cascade inactivates wild-type p53 in human HCC through the stabilization of the MDM4 protooncogene via a post-transcriptional mechanism involving an AKT-mediated phosphorylation of MDM4 and USP2a-mediated de-ubiquitination. Since the sustained activation of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis has impact on the survival probability of HCC patients, it may thus represent a promising therapeutic target. Citation Format: Rossella Pellegrino, Diego F. Calvisi, Olaf Neumann, Xin Chen, Chunmei Wang, Bernhard Radlwimmer, Sara Ladu, Frank Dombrowski, Matthias Evert, Peter Schirmacher, Thomas Longerich. The EEF1A2-PI3K-AKT-mTOR axis supports the protumorigenic function of MDM4 in human hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4300. doi:10.1158/1538-7445.AM2013-4300
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