Crosstalk between macrophage-derived PGE₂ and tumor UHRF1 drives hepatocellular carcinoma progression

Abstract

<b>Background:</b> Tumor-associated macrophages (TAMs) and dysregulated tumor epigenetics contribute to hepatocellular carcinoma (HCC) progression. However, the mechanistic interactions between TAMs and tumor epigenetics remain poorly understood. <b>Methods:</b> Immunohistochemistry and multiplexed fluorescence staining were performed to evaluate the correlation between TAMs numbers and UHRF1 expression in human HCC tissues. PGE₂ neutralizing antibody and COX-2 inhibitor were used to analyze the regulation of TAMs isolated from HCC tissues on UHRF1 expression. Multiple microRNA prediction programs were employed to identify microRNAs that target <i>UHRF1</i> 3'UTR. Luciferase reporter assay was applied to evaluate the regulation of miR-520d on UHRF1 expression. Chromatin immunoprecipitation (ChIP) assays were performed to assess the abundance of H3K9me2 in the <i>KLF6</i> promoter and DNMT1 in the <i>CSF1</i> promoter regulated by UHRF1. The functional roles of TAM-mediated oncogenic network in HCC progression were verified by <i>in vitro</i> colony formation assays, <i>in vivo</i> xenograft experiments and analysis of clinical samples. <b>Results:</b> Here, we find that TAMs induce and maintain high levels of HCC UHRF1, an oncogenic epigenetic regulator. Mechanistically, TAM-derived PGE₂ stimulates UHRF1 expression by repressing miR-520d that targets the 3'-UTR of <i>UHRF1</i> mRNA. In consequence, upregulated UHRF1 methylates H3K9 to diminish tumor KLF6 expression, a tumor inhibitory transcriptional factor that directly transcribes miR-520d. PGE₂ reduces KLF6 occupancy in the promoter of miR-520d, dampens miR-520d expression, and sustains robust UHRF1 expression. Moreover, UHRF1 promotes CSF1 expression by inducing DNA hypomethylation of the <i>CSF1</i> promoter and supports TAM accumulation. <b>Conclusions:</b> Capitalizing on studies on HCC cells and tissues, animal models, and clinical information, we reveal a previously unappreciated TAM-mediated oncogenic network via multiple reciprocal enforcing molecular nodes. Targeting this network may be an approach to treat HCC patients.