Human oncoprotein MDM2 activates the Akt signaling pathway through an interaction with the repressor element-1 silencing transcription factor conferring a survival advantage to cancer cells

Abstract

The current paradigm states that the Akt signaling pathway phosphorylates the human oncoprotein mouse double minute 2 (MDM2), leading to its nuclear translocation and degradation of the tumor suppressor p53. Here we report a novel Akt signaling pathway elicited by MDM2. Upregulation of endogenous MDM2 promotes, whereas its downregulation diminishes, Akt phosphorylation irrespective of p53 status. MDM2 requires phosphatidylinositol (PI)3-kinase activity for enhancing Akt phosphorylation and upregulates this activity by repressing transcription of the regulatory subunit p85 of PI3-kinase. MDM2 interacts with the repressor element-1 silencing transcription factor (REST), a tumor suppressor that functions by downregulating PI3-kinase activity and Akt phosphorylation, prevents localization of REST on the p85 promoter and represses p85 expression. The deletion mutant of MDM2 capable of upregulating Akt phosphorylation represses p85 expression and interferes with localization of REST on the p85 promoter, whereas the deletion mutant of MDM2 that does not increase Akt phosphorylation cannot perform these functions. Silencing of REST abrogates the ability of MDM2 to upregulate Akt phosphorylation and downregulate p85 expression, implicating the ability of MDM2 to interact with REST in its ability to inhibit p85 expression and activate Akt phosphorylation. Inhibition of MDM2-mediated Akt phosphorylation with an Aktphosphorylation-specific inhibitor abrogates its ability to improve cell survival. Consistently, the Akt phosphorylation function of MDM2 was required for its ability to improve cell survival after treatment with a chemotherapeutic drug. Our report not only unravels a novel signaling pathway that contributes to cell survival but also implicates a p53-independent transcription regulatory function of MDM2 in Akt signaling. The human homolog of the mouse double minute-2 gene product (MDM2) is frequently overexpressed in human sarcomas and carcinomas, and amplification of the mdm2 gene enhances the tumorigenic potential of murine cells, 1,2 implicating the genetic alteration in oncogenesis. MDM2 interacts with several growth suppressors, including the wild-type (WT) tumor suppressor p53, the retinoblastoma susceptibility gene product (Rb) and the growth suppressor p14.1,3 Although these interactions contribute to its oncogenic function, overexpression of MDM2 is thought to induce oncogenesis primarily by inactivating WT p53.MDM2 recognizes the transactivation domain of p53 and inactivates p53-mediated transcriptional activation; 4 it is also an E3 ubiquitin ligase and degrades p53 by targeting it for ubiquitination. 5 Small-molecule anti-MDM2 drugs, such as Nutlin or MDM2-specific silencer RNA, induce apoptosis and drug sensitivity in cell lines harboring WT p53 by elevating p53 levels. 6 Overexpression of MDM2 reduces the chemotherapeutic sensitivity of cancer cells by inactivating and degrading WT p53. 7Cancer cells with mutated p53 often overexpress MDM2, and the prognosis of this group of cancers is worse than th...

Mutant p53 establishes targetable tumor dependency by promoting unscheduled replication

“…Generation of plasmids, lentiviruses, and stable transfectants expressing GOF p53 or shRNA against GFP or p53 was carried out using pLKO.1 expression vector purchased from Open Biosystem following the supplier's protocols. Lung cells from mice were generated and cultured following standard protocols (28,62). CCNA2 promoter was a gift from Toshio Nikaido (63).…”

Constitutive Activation of DNA Damage Checkpoint Signaling Contributes to Mutant p53 Accumulation via Modulation of p53 Ubiquitination

“…WI38 (WT p53) normal lung fibroblasts were purchased from ATCC and maintained in Eagle’s MEM (ATCC) plus 10% FBS. ABC1 cells (P278S) (adenocarcinoma) expressing shGFP or shMDM2 (18) were maintained in MEM plus 10% FBS and (1 ug/ml) puromycin (Gibco). H1793, H1975 (both R273H; NSCLC), MDA MB 231 (R280K; breast), U2OS (WT p53; osteosarcoma), and 293T cells (WT p53; human embryonic kidney) were purchased from ATCC and maintained in DMEM plus 10% FBS.…”

The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing

“…Generation of plasmids and lentiviral vectors (pLKO.1) expressing short hairpin (sh) RNA against MDM2 or non-endogenous green fluorescence protein (GFP) from U6 promoter and harboring a puromycin resistance gene has been described earlier (20,29). …”