Abstract
BackgroundMDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA.Methodology/Principal FindingsHere we report that MDM4 mRNA is a target of hsa-mir-34a (miR-34a). MDM4 mRNA contains a lengthy 3′ untranslated region; however, we find that it is a miR-34a site within the open reading frame (ORF) of exon 11 that is responsible for the repression. Overexpression of miR-34a, but not a mutant miR-34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR-34a target genes. Likewise, MDM4 protein levels are decreased by miR-34a overexpression. Inhibition of endogenous miR-34a increased expression of miR-34a target genes and MDM4. A portion of MDM4 exon 11 containing this 8mer-A1 miR-34a site fused to a luciferase reporter gene is sufficient to confer responsiveness, being inhibited by additional expression of exogenous mir-34a and activated by inhibition of miR-34a.Conclusions/SignificanceThese data establish a mechanism for the observed DNA damage-induced negative regulation of MDM4 and potentially provide a novel means to manipulate MDM4 expression without introducing DNA damage.
Highlights
The gene MDM4 has become a target of interest for therapeutic intervention in cancer
The frequency of MDM4 overexpression varies by cancer type; for example, a majority of retinoblastomas show amplification of MDM4 [6], but it is very uncommon in prostate cancer [26]
In order to explore the correlation between MDM4 expression and miR-34a expression, we surveyed a variety of cell lines for expression of each (Figure 1)
Summary
The gene MDM4 has become a target of interest for therapeutic intervention in cancer. MDM4 serves as an important negative regulator of the p53 tumor suppressor. The importance of MDM4 in human cancer is underscored by its frequent amplification in certain tumor types, such as colon cancer [2], gliomas [3,4,5] and retinoblastomas [6]. Full activation of p53 in response to DNA damage requires inhibition of MDM4 [7]. MDM4, called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA
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