doi: 10.3390/cancers13020199.
Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC
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- PMID:33429878
- PMCID: PMC7829828
- DOI: 10.3390/cancers13020199
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Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC
Rossella Pellegrino et al. Cancers (Basel)..
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Abstract
Different molecular mechanisms support the overexpression of the mouse double minute homolog 4 (MDM4), a functional p53 inhibitor, in human hepatocellular carcinoma (HCC). However, the transcription factors (TFs) leading to its transcriptional upregulation remain unknown. Following promoter and gene expression analyses, putative TFs were investigated using gene-specific siRNAs, cDNAs, luciferase reporter assays, chromatin immunoprecipitation, and XI-011 drug treatment in vitro. Additionally, MDM4 expression was investigated inSRF-VP16iHep transgenic mice. We observed a copy-number-independent upregulation ofMDM4 in human HCCs. Serum response factor (SRF), ELK1 and ELK4 were identified as TFs activatingMDM4 transcription. While SRF was constitutively detected in TF complexes at theMDM4 promoter, presence of ELK1 and ELK4 was cell-type dependent. Furthermore, MDM4 was upregulated in SRF-VP16-driven murine liver tumors. The pharmacological inhibitor XI-011 exhibited anti-MDM4 activity by downregulating the TFs drivingMDM4 transcription, which decreased HCC cell viability and increased apoptosis. In conclusion, SRF drives transcriptionalMDM4 upregulation in HCC, acting in concert with either ELK1 or ELK4. The transcriptional regulation ofMDM4 may be a promising target for precision oncology of human HCC, as XI-011 treatment exerts anti-MDM4 activity independent from theMDM4 copy number and thep53 status.
Keywords: ELK1; ELK4; ERK; ETS transcription factors; HCC; MDM4; MDM4 transcriptional regulation; SRF; XI-011; tumor protein p53.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Aberrant transcriptional activation may be involved in upregulation of mouse double minute homolog 4 (MDM4) in human HCC. (A) RelativeMDM4 mRNA expression in human hepatocellular carcinomas (HCCs) with balanced (n = 13) and gainedMDM4 (n = 24) gene loci, respectively. Mann–Whitney U test:p > 0.05. (B) An in silico analysis of the basalMDM4 promoter region identified putative transcription factor binding sites for serum response factor (SRF), ELK1, and ELK4. (C) Expression profiling revealed a positive association betweenMDM4 mRNA and the expression level of the putative transcription factorsSRF,ELK1, andELK4 in human HCC samples (n = 37). (D)MDM4,SRF, andELK4 mRNA levels were associated with the survival probability of HCC patients in a second cohort (n = 32). Each median expression level was used for stratification. Abbreviation: n.s., not statistically significant.
SRF, ELK1, and ELK4 regulate MDM4 expression in HCC cell lines. (A) IncreasedMDM4 mRNA levels in HepG2 and HLE cell lines following fetal calf serum (FCS) stimulation compared to starved control cells. (B) MDM4 mRNA and (C) protein levels following siRNA-mediated knockdown of SRF compared to control cells transfected with a scrambled, nonsense siRNA (siNS) in HepG2 and HLE cells. (D) MDM4 mRNA and (E) protein levels following siRNA-mediated knockdown of ELK1 compared to control cells transfected with a scrambled, nonsense siRNA (siNS) in HepG2 and HLE cells. (F) MDM4 mRNA and (G) protein levels following siRNA-mediated knockdown of ELK4 compared to control cells transfected with a scrambled, nonsense siRNA (siNS) in HepG2 and HLE cells. (H) siRNA-mediated knockdown of SRF (siSRF_2) prevents FCS-stimulated MDM4 protein upregulation. (I) MDM4 mRNA and protein expression 48 h following transfection of HuH7 cells with an SRF-VP16 expression vector compared to mock transfected control cells. Original western blots are shown in Figures S7 and S8. Data are presented as mean ± SEM. Mann–Whitney U test: *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: siNS—scrambled, nonsense siRNA; siSRF_1/_2, siELK1_1/_2, siELK4_1/_2—siRNA 1 and 2 specifically targeting SRF, ELK1, and ELK4, respectively.
ELK1 and ELK4 are essential co-factors for SRF-mediated transcriptional regulation ofMDM4 in HCC. Luciferase activity of aMDM4 promoter reporter upon siRNA-mediated knockdown of (A) SRF, (B) ELK1, and (C) ELK4 in HepG2 and HLE cells compared to controls. (D)MDM4 mRNA levels after co-transfection of anELK1 cDNA with siNS or siSRF. Transfection efficacy was confirmed by detection ofELK1 andSRF mRNA levels. (E)MDM4 mRNA levels following transfection of the indicated cDNA plasmids. ELK1 S383A represents an inactive variant, which cannot be activated by phosphorylation of S383 and is thus unable to initiate target gene transcription. Transfection efficacy was confirmed by detection ofELK1 andSRF mRNA levels. Data are presented as mean ± SEM. Mann-Whitney U test: *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: siNS, scrambled, nonsense; siSRF_1/_2, siELK1_1/_2 siELK4_1/_2, siRNA 1 and 2 specifically targeting SRF, ELK1 and ELK4, respectively; ELK1, ELK1 cDNA; ELK1 S383A, ELK1 S383A cDNA; GLuc,Gaussia luciferase; SEAP, Secreted Alkaline Phosphatase; norm., normalized against control.
MDM4 gene promoter is activated by an SRF-ETS family transcription factor complex in HCC cell lines. (A) Schematic representation of the positioning of primers used for ChIP analyses of theMDM4 promoter region. (B) Specific binding of SRF, ELK1, and ELK4 at their cognate binding sites in theMDM4 promoter compared to control primers located either down- or upstream of the predicted basalMDM4 promoter as detected by quantitative real-time PCR of immunoprecipitated chromatin. (C) Relative enrichment of SRF-immunoprecipitated DNA in HuH7 cells transfected with SRF-VP16 expression plasmid compared to mock transfected control cells. Data are presented as mean ± SEM. Mann–Whitney U test: **p < 0.01, ***p < 0.001. Abbreviations: upstr. control, control primer amplifying a region upstream of theMDM4 promoter; downstr. control, control primer amplifying a region downstream of theMDM4 promoter.
MDM4 is upregulated inSRF-VP16iHep transgenic mice. (A) Normal liver parenchyma in control mice (HE staining). (B) Well-differentiated HCC in a 30-week-oldSRF-VP16iHep mouse showing trabecular disarray and pseudogland formation. MDM4 immunostaining is negative in control mice (C), while a diffuse, predominantly nuclear staining is seen inSRF-VP16iHep mice (D). Individual hepatocyte nuclei (arrow) are positive for phosphorylated-ELK1 in the control liver (E), whereas the number of p-ELK1 positive nuclei is significantly increased inSRF-VP16iHep mice (F). There is no ELK4 immunosignal in control mice (G). In contrast, the SRF-VP16-induced HCC reveals weak to moderate nuclear ELK4 staining (H). Scale bar: 20 µM.
XI-011 inhibitsMDM4 transcription by transcription factor downregulation. (A)MDM4 mRNA level at 16 h following XI-011 treatment of HepG2 and HLE cells. (B) MDM4 protein expression as detected by Western immunoblots and corresponding densitometric analysis (lower panel) 16 h following XI-011 treatment of HepG2 and HLE cell lines. (C) Luciferase activity of aMDM4 promoter reporter following XI-011 treatment in HepG2 and HLE cells, respectively. (D) Induction of p53 and PARP protein cleavage following XI-011 treatment of HCC cell lines as indicated. (E) XI-011 treatment led top21 mRNA induction in both HCC cell lines. (F) p53 protein expression over time following XI-011 treatment (1 µM) with or without additional cycloheximide (CHX) treatment inp53-wildtype HepG2 andp53-mutant HLE cells as detected by Western immunoblotting. (G) Relative cell viability of HepG2 and HLE cells over time after XI-011 treatment using the indicated doses compared to control cells. (H) Western immunoblots following XI-011 treatment of HepG2 and HLE cells. Original western blots are shown in Figures S9–S12. Data are presented as mean ± SEM. One-way and two-way ANOVA with Tukey’s test were used in panel C and G, respectively; all the other data were analyzed by Kruskal–Wallis followed by Dunn’s test: *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: fl, full-length PARP protein; cl, cleaved PARP protein; GLuc,Gaussia luciferase; SEAP, secreted alkaline phosphatase; norm., normalized against control; CHX, cycloheximide.
References
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