MDMX Regulates Transcriptional Activity of p53 and FOXO Proteins to Stimulate Proliferation of Melanoma Cells

SIMPLE SUMMARY: We have investigated the transcriptional changes occurring in uveal and cutaneous melanoma cell lines upon depletion of MDMX (aka:MDM4). Computational analyses of the mRNAs/genes affected upon MDMX depletion determined that many were containing a p53-bindingsite, but even more contained a FOX recognition site(s). Since connections between MDM2 and FOXO1 had already been published, we investigated whether indeed a subset of the MDMX-regulated genes are dependent on FOXO1/FOXO3 expression. Indeed, a number of such target genes, i.e., PIK3IP1, MXD4 and ZMAT3, were found to be FOXO target genes in our cell models. Some of these genes were recently identified as indirect p53-target genes, and their expression was found to be regulated by RFX7 transcription factor, which was found activated upon pharmacological activation of p53, e.g., by Nutlin-3. However, a clear involvement of RFX7 in our model could not be established, but an interplay between FOXO and RFX7 factors seems evident. ABSTRACT: The tumor suppressor protein p53 has an important role in cell-fate determination. In cancer cells, the activity of p53 is frequently repressed by high levels of MDMX and/or MDM2. MDM2 is a ubiquitin ligase whose activity results in ubiquitin- and proteasome-dependent p53 degradation, while MDMX inhibits p53-activated transcription by shielding the p53 transactivation domain. Interestingly, the oncogenic functions of MDMX appear to be more wide-spread than inhibition of p53. The present study aimed to elucidate the MDMX-controlled transcriptome. Therefore, we depleted MDMX with four distinct shRNAs from a high MDMX expressing uveal melanoma cell line and determined the effect on the transcriptome by RNAseq. Biological function analyses indicate the inhibition of the cell cycle regulatory genes and stimulation of cell death activating genes upon MDMX depletion. Although the inhibition of p53 activity clearly contributes to the transcription regulation controlled by MDMX, it appeared that the transcriptional regulation of multiple genes did not only rely on p53 expression. Analysis of gene regulatory networks indicated a role for Forkhead box (FOX) transcription factors. Depletion of FOXO proteins partly prevented the transcriptional changes upon MDMX depletion. Furthermore, depletion of FOXO proteins relatively diminished the growth inhibition upon MDMX knockdown, although the knockdown of the FOXO transcription factors also reduces cell growth. In conclusion, the p53-independent oncogenic functions of MDMX could be partially explained by its regulation of FOXO activity.