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

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.

Keywords: FOXO; MDM2; MDMX; RFX7; RNA-seq; cutaneous melanoma; p53; uveal melanoma.

Figure 1

Kinetics of p53 activation upon MDMX depletion in MEL202 cells. (A) Protein expression analysis of i-shCtrl and i-shMDMX MEL202 cells harvested after different incubation periods with doxycycline (0, 24, 48 and 72 h). The cells containing the distinct MDMX targeting shRNA constructs (#1, 2, 3 and 4) show a clear reduction of MDMX protein upon doxycycline treatment (10 ng/mL). Simultaneously with MDMX depletion, p53 levels slightly increase and p21^CIP1 levels rise, mostly at the later time points. (B) Normalized, relative mRNA expression of CDKN1A and MAD2L1 in i-shCtrl and i-shMDMX MEL202 cells harvested after different incubation periods with doxycycline (D; 10 ng/mL) or Vehicle (V). Expression of CDKN1A is markedly increased upon MDMX depletion already after 24 h and only slightly increases at later time points. Repression of MAD2L1 upon MDMX depletion takes approximately 48 h before reaching a plateau. Significant alterations (p < 0.05) in expression levels are indicated with *. Original blots see Supplementary File S1.

Figure 2

Verification of MDMX target genes. (A) Relative, normalized mRNA expression of CENPF, KIF23, PIK3IP1 and PTCHD4 genes after 48 h of doxycycline (D 10 ng/mL) or Vehicle (V) treatment of indicated cell lines. CENPF and KIF23 are downregulated and PIK3IP1 and PTCHD4 are upregulated upon MDMX knockdown. Significant alterations (p < 0.05) in expression levels are indicated with *. (B) Analysis of protein expression after 48 h doxycycline treatment shows a consistent repression of MAD2L1 level and an increase in p53, FOXO1, p21 and PUMA levels upon MDMX depletion. (C,D) MEL202/CR-Ctrl and MEL202/CR-p53 cells were depleted for MDMX (48 h doxycycline (D), 10 ng/mL) or control-treated (V) after which RNA and protein lysates were harvested and analysed by qRT-PCR (C) and Western blotting (D). Significant alterations (p < 0.05) in expression levels are indicated with *. Original blots see Supplementary File S1.

Figure 2

Verification of MDMX target genes. (A) Relative, normalized mRNA expression of CENPF, KIF23, PIK3IP1 and PTCHD4 genes after 48 h of doxycycline (D 10 ng/mL) or Vehicle (V) treatment of indicated cell lines. CENPF and KIF23 are downregulated and PIK3IP1 and PTCHD4 are upregulated upon MDMX knockdown. Significant alterations (p < 0.05) in expression levels are indicated with *. (B) Analysis of protein expression after 48 h doxycycline treatment shows a consistent repression of MAD2L1 level and an increase in p53, FOXO1, p21 and PUMA levels upon MDMX depletion. (C,D) MEL202/CR-Ctrl and MEL202/CR-p53 cells were depleted for MDMX (48 h doxycycline (D), 10 ng/mL) or control-treated (V) after which RNA and protein lysates were harvested and analysed by qRT-PCR (C) and Western blotting (D). Significant alterations (p < 0.05) in expression levels are indicated with *. Original blots see Supplementary File S1.

Figure 3

Expression of some MDMX target genes is FOXO-dependent. Analyses of MEL202/CR-Ctrl and MEL202/CRp53 cell lines, with and without 2 distinct i-shMDMX shRNAs, with and without i-shFOXO, plus and minus doxycycline treatment. (A) Indicated cell lines were treated for 72 h with 10 ng/mL doxycycline after which protein lysates were harvested and expression of designated proteins was analyzed to assess the efficacy of depletion. (B) The various cell lines were treated for 72 h with 10 ng/mL doxycycline (D) or Vehicle (V) after which RNA was harvested and the expression of indicated genes was analyzed by real-time qPCR. Significant alterations (p < 0.05) in expression levels are indicated with *. Original blots see Supplementary File S1.

Figure 4

MDMX stimulates growth of UM cell lines MEL202 via attenuation of p53 and FOXO activity. (A) Indicated MEL202-derived cell lines (CR-Ctrl and CR-p53; i-shCtrl or i-shFOXO) either containing an i-shCtrl or two distinct i-shMDMX shRNA constructs, were treated with doxycycline (D; 10 ng/mL) for 5 days. Relative survival of the cell lines, each normalized to Vehicle (V)-treated samples. Significant alterations (p < 0.05) in cell survival are indicated with *. (B) Relative survival of the indicated cell lines but now normalized to i-shCtrl + Doxycycline to determine the effect of MDMX depletion in i-shCtrl versus i-shFOXO cells. Significant alterations (p < 0.05) in cell survival are indicated with *. (C) Indicated MEL202-derived cell lines (CR-Ctrl and CR-p53; i-shCtrl or i-shFOXO) either containing and i-shCtrl or i-shMDMX shRNA construct were treated with doxycycline (D; 10 ng/mL) for 3 days. Relative survival of the cell lines all normalized to Vehicle (V)-treated samples. Significant alterations (p < 0.05) in cell survival are indicated with *. (D) Relative survival of the indicated cell lines but now normalized to i-shCtrl + Doxycycline to determine the effect of MDMX depletion in i-shCtrl versus i-shFOXO cells. Significant alterations (p < 0.05) in cell survival are indicated with *.

Figure 5

Analyses of RFX7 involvement in transcriptional regulation of genes upregulated upon MDMX depletion. (A,B) MEL202/CR-Ctrl and/CR-p53, i-shCtrl and i-shF1F3, containing either i-shCtrl or i-shMDMX shRNAs, were treated for 72 h with 10 ng/mL doxycycline (D) or Vehicle (V) after which RNA and protein was harvested and analyzed for the mRNA expression of RFX7 and the reported RFX7-target genes RFX5, PNRC1 and PDCD4 (A) and to evaluate the effect of MDMX depletion on RFX7 protein migration. Significant alterations (p < 0.05) in gene expression are indicated with *. These are the same samples as analyzed in Figure 3. (C) 92.1/i-shCtrl and 92.1/i-shF1F3, i-shCtrl and i-shMDMX cell lines were treated for 72 h with 10 ng/mL doxycycline, after which protein lysates were harvested and analyzed for RFX7 expression. These are the same samples as analyzed in Supplementary Figure S6. Original blots see Supplementary File S1.