High Mdm4 levels suppress p53 activity and enhance its half-life in acute myeloid leukaemia.
ABSTRACT: Although p53 is found mutated in almost 50% of all cancers, p53 mutations in leukaemia are relatively rare. Acute myeloid leukaemia (AML) cells employ other strategies to inactivate their wild type p53 (WTp53), like the overexpression of the p53 negative regulators Mdm2 and Mdm4. As such, AMLs are excellent candidates for therapeutics involving the reactivation of their WTp53 to restrict and destroy cancer cells, and the Mdm2 antagonist nutlin-3 is one such promising agent. Using AML cell lines with WTp53, we identified stable and high levels of p53 in the OCI/AML-2 cell lines. We demonstrate that this nutlin-3 sensitive cell line overexpressed Mdm4 to sequester, stabilise and inhibit p53 in the cytoplasm. We also show that elevated Mdm4 competed with Mdm2-p53 interaction and therefore extended p53 half-life while preventing p53 transcriptional activity. Our results provide biochemical evidence on the dynamics of the p53-Mdm2-Mdm4 interactions in affecting p53 levels and activity, and unlike previously reported findings derived from genetically manipulated systems, AML cells with naturally high levels of Mdm4 remain sensitive to nutlin treatment.Endogenously high levels of Mdm4 inhibit and sequester p53 in AML. High levels of Mdm4 do not block function of Mdm2 inhibitors in AML.
Project description:The purpose of the present study was to investigate the prognostic significance of murine double minute 4 (MDM4) in chronic lymphocytic leukemia (CLL) and to characterize the role of MDM4 in the p53 pathway. Full-length MDM4 (FL-MDM4), a splicing variant of MDM4 (S-MDM4) and murine double minute 2 (MDM2) mRNA expressions were detected by quantitative PCR in 140 Chinese patients with CLL, and primary CLL cells were treated in vitro with either fludarabine or Nutlin-3 to explore the interaction between p53 status and MDM4 or MDM2 expression. A marked increase of FL-MDM4 and S-MDM4 expressions were observed in the CLL patients with p53 aberrations (deletion and/or mutation) (P = 0.024, P < 0.001). A high level of S-MDM4 mRNA expression was associated with short treatment free survival (TFS) (P = 0.004). FL-MDM4 expression was significantly decreased after fludarabine treatment (P = 0.001) but increased after Nutlin-3 treatment (P = 0.008) of primary CLL cells without p53 aberrations. Both S-MDM4 and MDM2 expressions were significantly increased after fludarabine treatment of CLL cells without p53 aberrations (P = 0.013 and P = 0.030). MDM2 overexpression also occurred in CLL cells with p53 wild type after Nutlin-3 treatment (P = 0.018). FL-MDM4 and S-MDM4 overexpression are indicators of p53 aberrations in CLL patients, suggesting that those patients have a poor prognosis. FL-MDM4 inhibitory effects on p53 can be removed by MDM2-p53 and saved by Nutlin-3.
Project description:The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.
Project description:Murine double minute homolog 2 (MDM2) is an oncoprotein that induces p53 degradation via ubiquitin-ligase activity. MDM4 cooperates with MDM2-mediated p53 degradation, directly inhibiting p53 transcription by binding to its transactivation domain. Our previous study reported that the simultaneous inhibition of MDM2 and MDM4 using nutlin-3 (an inhibitor of the MDM2-p53 interaction) and chimeric small interfering RNA with DNA-substituted seed arms (named chiMDM2 and chiMDM4) more potently activated p53 than the MDM2 or MDM4 inhibitor alone and synergistically augmented antitumor effects in various types of cancer cells with the wild-type (wt) TP53. Recently, the synergism of MDM2 and mitogen-activated protein kinase kinase (MEK) inhibitors has been demonstrated in wt TP53 colorectal and non-small cell lung cancer cells harboring mutant-type (mt) KRAS. The current study examined whether chiMDM4 augmented the synergistic antitumor effects of MDM2 and MEK inhibition using chiMDM2 or nutlin-3 and trametinib, respectively. ChiMDM2 and trametinib used in combination demonstrated a synergistic antitumor activity in HCT116 and LoVo colon cancer cells, and SNU-1 gastric cancer cells harboring wt TP53 and mt KRAS. Furthermore, chiMDM4 synergistically enhanced this combinational effect. Similar results were observed when nutlin-3 was used instead of chiMDM2. MDM4/MDM2 double knockdown combined with trametinib treatment enhanced G1 arrest and apoptosis induction. This was associated with the accumulation of p53, suppression of phosphorylated-extracellular signal-regulated kinase 2, inhibition of retinoblastoma phosphorylation, suppression of E2F1-activated proteins, and potent activation of pro-apoptotic proteins, such as Fas and p53 upregulated modulator of apoptosis. The results inidcated that the triple inhibition of MDM4, MDM2 and MEK exerted a potent antitumor effect in wt TP53 colon and gastric cancer cells with mt KRAS. Simultaneous activation of p53 and inhibition of aberrant KRAS signaling may be a rational treatment strategy for gastrointestinal tumors.
Project description:The p53 tumor suppressor is mutated in most human tumors. MDM2, a well-known inhibitor of p53, is overexpressed in a large number of tumors, suggesting that increased levels of MDM2 also contribute to tumorigenesis. A novel p53 inhibitor, MDM4, was more recently identified. The role of MDM4 in cancer development is not well understood. We set out to examine the levels of MDM4 by immunohistochemistry in head and neck squamous carcinomas (HNSC) to ask whether high MDM4 levels could contribute to its development and progression. In addition, MDM2 and p53 levels were examined to identify overlapping expression patterns. MDM4 is present at high levels in 50% of HNSC. In addition, overexpression of MDM2 was detected in 80% of tumors, many of which were also positive for MDM4. A subset of tumors displayed high levels of all 3 proteins. Sequencing of the p53 gene revealed that tumors with positive immunoreactivity for MDM2 or MDM4, some of which also had high levels of p53, did not carry mutations in this gene. Thus, the detection of p53 by immunohistochemistry was not synonymous with the presence of p53 mutations. Expression of both MDM2 and MDM4 in tumors without p53 mutations strongly suggests that MDM2 and MDM4 inhibit the activity of this tumor suppressor in HNSC.
Project description:The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status.
Project description:The Mdm2 and Mdm4 oncoproteins are key negative regulators of the p53 tumor suppressor. However, their physiological contributions to the regulation of p53 stability and activity remain highly controversial. Here, we combined a p53 knock-in allele, in which p53 is silenced by a transcriptional stop element flanked by loxP sites, with the mdm2- and mdm4-null alleles. This approach allows Cre-mediated conditional p53 expression in tissues in vivo and cells in vitro lacking Mdm2, Mdm4, or both. Using this strategy, we show that Mdm2 and Mdm4 are essential in a nonredundant manner for preventing p53 activity in the same cell type, irrespective of the proliferation/differentiation status of the cells. Although Mdm2 prevents accumulation of the p53 protein, Mdm4 contributes to the overall inhibition of p53 activity independent of Mdm2. We propose a model in which Mdm2 is critical for the regulation of p53 levels and Mdm4 is critical for the fine-tuning of p53 transcriptional activity, both proteins acting synergistically to keep p53 in check.
Project description:Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically.
Project description:The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development.
Project description:The proteins MDM2 and MDM4 are key negative regulators of the tumor suppressor protein p53, which are frequently upregulated in cancer cells. They inhibit the transactivation activity of p53 by binding separately or in concert to its transactivation domain. MDM2 is also a ubiquitin ligase that leads to the degradation of p53. Accordingly, MDM2 and MDM4 are important targets for drugs to inhibit their binding to p53. We found from in silico screening and confirmed by experiment that lithocholic acid (LCA) binds to the p53 binding sites of both MDM2 and MDM4 with a fivefold preference for MDM4. LCA is an endogenous steroidal bile acid, variously reported to have both carcinogenic and apoptotic activities. The comparison of LCA effects on apoptosis in HCT116 p53(+/+) vs. p53(-/-) cells shows a predominantly p53-mediated induction of caspase-3/7. The dissociation constants are in the ?M region, but only modest inhibition of binding of MDM2 and MDM4 is required to negate their upregulation because they have to compete with transcriptional coactivator p300 for binding to p53. Binding was weakened by structural changes in LCA, and so it may be a natural ligand of MDM2 and MDM4, raising the possibility that MDM proteins may be sensors for specific steroids.
Project description:A wild-type (wt) p53 gene characterizes thyroid tumors, except for the rare anaplastic histotype. Because p53 inactivation is a prerequisite for tumor development, alterations of p53 regulators represent an alternative way to impair p53 function. Indeed, murine double minute 2 (MDM2), the main p53 negative regulator, is overexpressed in many tumor histotypes including those of the thyroid. A new p53 regulator, MDM4 (a.k.a. MDMX or HDMX) an analog of MDM2, represents a new oncogene although its impact on tumor properties remains largely unexplored. We estimated levels of MDM2, MDM4, and its variants, MDM4-S (originally HDMX-S) and MDM4-211 (originally HDMX211), in a group of 57 papillary thyroid carcinomas (PTC), characterized by wt tumor protein 53, in comparison to matched contra-lateral lobe normal tissue. Further, we evaluated the association between expression levels of these genes and the histopathological features of tumors. Quantitative real-time polymerase chain reaction revealed a highly significant downregulation of MDM4 mRNA in tumor tissue compared to control tissue (P<0.0001), a finding confirmed by western blot on a subset of 20 tissue pairs. Moreover, the tumor-to-normal ratio of MDM4 levels for each individual was significantly lower in late tumor stages, suggesting a specific downregulation of MDM4 expression with tumor progression. In comparison, MDM2 messenger RNA (mRNA) and protein levels were frequently upregulated with no correlation with MDM4 levels. Lastly, we frequently detected overexpression of MDM4-S mRNA and presence of the aberrant form, MDM4-211 in this tumor group. These findings indicate that MDM4 alterations are a frequent event in PTC. It is worthy to note that the significant downregulation of full-length MDM4 in PTC reveals a novel status of this factor in human cancer that counsels careful evaluation of its role in human tumorigenesis and of its potential as therapeutic target.