Combining p53-activating agents with HDAC inhibition results in suppression of FOXM1 and other genes associated with poor prognosis prostate cancer


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Gemma Gregg1,Fiammetta Falcone1,Andrea Lees1,Gerard Quinn1,Richard Kennedy2,Ian Mills1,Simon McDade1
1Queens University Belfast,2Almac Diagnostics

Abstract

Background

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men worldwide. SOC combines radiotherapy with ADT, however recurrence rate remains unacceptably high and many men present with late-stage aggressive and metastatic disease.

Despite p53 being mutated in 50% of tumours, it has been reported that only 12% of primary PCa harbour a p53 alteration. Emerging data links epigenetic-mediated gene silencing as a differentiator between aggressive and indolent disease, associated with up-regulation of cell cycle regulator FOXM1- also known to be suppressed by p53. HDACi are an emerging class of epigenetic modifying anti-cancer agents that we hypothesise will enhance p53 mediated cell deathwhilst also altering patterns of gene expression totarget p53-dependent and independent vulnerabilities in PCa.

Method

We used basally p53 deficient/proficient PCa models (PC3/LNCaP) and novel C4-2B and LNCaP p53 CRISPR-Cas9 knockout models to investigate the combination effects of Nutlin 3A or radiation (IR) with class 1 HDACi Entinostat. We did this by Annexin V/PI Flow Cytometry and Western Blot. RT-PCR and RNA-seq were utilised to evaluate transcriptional effects. TCGA mutational and RNA-seq data were used to link p53 mutation with altered gene expression and patient outcome.

Results

We have demonstrated a p53-dependent synergistic induction of cell death when we combine N3A/IR with Entinostat. In addition, we see that p53 acts as a barrier to genes associated with poor prognosis- such as FOXM1- which is phenocopied by single-agent Entinostat independent of WT-p53. This suppression may potentially be downstream of p21 via the DREAM complex. Importantly p53 mutation in TCGA primary PCa correlated with elevated FOXM1 expression and poorer patient outcome.

Conclusion

Results to date suggest that Entinostat enhances p53-dependent cell death but also has additional p53-dependent and independent mechanisms to suppress FOXM1. This mechanism looks to be dependent on p21 and may involve DREAM complex.