p53-R172H cooperates with Brd4 to induce Csf-1 expression and lung metastasis: H3K27ac profile of mouse ESCC cells with Trp53R172/- or Trp53+/+

TP53 mutations are frequent in esophageal squamous cell carcinoma (ESCC), and other SCCs, and are associated with a proclivity for metastasis. Here, we report that the Colony-Stimulating Factor-1 (CSF-1) expression is upregulated significantly in a p53-R175H (R172H in mice)-mediated manner in lung metastatic lesions of ESCC. The p53-R172H-dependent Csf-1 signaling through its cognate receptor Csf-1r increases tumor cell invasion and lung metastasis, which is mediated in part through the phosphorylation of Stat3 and epithelial-to-mesenchymal transition. In Trp53R172H tumor cells, p53 binds to the Csf-1 promoter and the Csf-1 locus is enriched with H3K27ac, which likely is permissive for fostering an interaction between Brd4 and p53-R172H to regulate the Csf-1 transcription. Inhibition of Brd4 not only reduces tumor invasion and lung metastasis but also reduces the Csf-1 level in circulation. Overall, our results establish that the p53-R175H-mediated Brd4-Csf-1 axis promotes ESCC lung metastasis and suggest avenues for therapeutic strategies for this difficult to treat disease. To elucidate the mechanism for how Trp53R172H regulates Csf-1 transcription, we conducted Cleavage Under Targets and Release Using Nuclease sequencing (CUT&RUN-seq) for histone 3 lysine 27 acetylation (H3K27ac), which is an active enhancer and promoter marker [48], on Trp53R172H/- or Trp53+/+ ESCC cells. The genome-wide analysis revealed 1189 genes having enriched and 1454 genes having depleted H3K27ac in Trp53R172H/- tumor cells compared to Trp53+/+ cells (|log2fold change| > 1.5). Notably, H3K27ac peaks were enriched across Vim gene encoding the mesenchymal marker Vimentin and were reduced across Cdh1 gene encoding the epithelial marker E- Cadherin in Trp53R172H tumor cells compared to Trp53+/+ cells. Interestingly, H3K27ac peaks were enriched across Csf-1 locus including the promoter site in Trp53R172H/- ESCC cells compared to Trp53+/+ tumor cells, suggesting that the Csf-1 locus was accessible and active in the presence of p53-R172H. The epigenetic reader Brd4 binds to acetylated histone lysine residues at promoters and super-enhancers to regulate transcription initiation [29]. We therefore investigated if Brd4 was recruited to enriched H3K27ac sites in ESCC cells to upregulate Csf-1. We found that global Brd4 level in Trp53R172H/- tumor cells was ~2-fold higher than in Trp53-/- cells and ~3-fold higher than in Trp53+/+ cells suggesting gain-of-function properties of p53-R172.