The interaction of Serum Response Factor (SRF) and ETS domain transcription factors regulate the transcription of the MDM 4 oncogene in HCC

The Mouse Double Minute 4 protein (MDM 4) is a p53-negative regulator, which inhibits its transcriptional activity. As previously demonstrated, MDM 4 mRNA and protein levels are upregulated in human hepatocellular carcinoma (HCC) as consequence of both copy number alterations as well as a post-transcriptional mechanism involving the AKT/mTOR axis. However, many human HCCs showed high MDM 4 mRNA levels with a balanced MDM 4 gene locus, leading us to hypothesize that aberrant transcriptional mechanism might be involved in the upregulation of MDM 4 in human HCC.

To identify transcription factors (TFs) involved in the regulation of MDM 4 transcription, in silico promoter analysis was performed using the Jaspar and Mapper databases. Profiling data of human HCCs were assessed for a potential association between the expression levels of candidate TFs and MDM 4 mRNA, which was complemented by a survival analysis. Transfection of gene-specific siRNAs, cDNA constructs and luciferase promoter reporters as well as chromatin immunoprecipitation (ChIP) were used to validate the putative TFs responsible for the transcriptional regulation of MDM 4 in human HCC cell lines in vitro. These data were corroborated by in vivo analyses using a SRF-VP16 transgenic HCC mouse model. Finally, XI-011, a pseudourea derivate, was used for pharmacological targeting of MDM 4 transcription in vitro.

Overexpression of MDM 4 was observed in human HCC independently from its gene copy number. In silico analysis suggested SRF, ELK1 and ELK4 may be putative TFs binding to the MDM 4 promoter region. A strong positive correlation between SRF and MDM 4 expression was observed in a human HCC samples and high mRNA levels of MDM 4, SRF, and ELK4 were associated with a shorter survival probability of HCC patients following liver resection. In line, inhibition of the individual TFs by gene-specific siRNAs significantly decreased the MDM 4 mRNA levels, the most prominent effect exerted by targeting of SRF. Similarly, ELK1 or ELK4 depletion decreased the luciferase activity of a MDM 4 promoter luciferase reporter in HCC cell lines. Additionally, transient overexpression of SRF or ELK1 increased MDM 4 expression in HuH7 cells; however, transfection of an ELK1 cDNA plasmid together with gene-specific siRNAs against SRF did not rescue the MDM 4 mRNA levels in HLE cells compared to cells overexpressing ELK1 alone, demonstrating that SRF is the main driver of MDM 4 transcription. Moreover, ChIP analyses revealed an enrichment of binding SRF and its co-factors to the MDM 4 core promoter in vitro and in vivo. More interestingly, XI-011 treatment of HepG2 and HLE cells led to downregulation of MDM 4 mRNA levels by decreasing SRF, ELK1, and ELK4 protein levels, which resulted in reduced cell viability due to the induction of apoptosis in vitro.

Taken together, our data demonstrated a crucial role for SRF and its transcriptional cofactors in supporting the oncogenic function of MDM 4 in HCC. Considering that most human HCCs show overexpression of MDM 4 independently from its gene copy number, targeting the MDM 4 transcriptional machinery may represent a promising therapeutic approach for liver cancer patients.