H3K27me3-mediated regulation of PD-L1 expression in triple-negative breast cancer (TNBC)

Purpose: Enhancer of zeste homolog 2 (EZH2) is highly expressed in triple-negative breast cancer (TNBC) and induces massive histone modification via trimethylation at lysine 27 of histone H3 (H3K27me3). The expression level of programmed death ligand 1 (PD-L1) is crucial for determining the indications for immune checkpoint inhibitors in patients with TNBC. This study aimed to clarify the regulatory roles of EZH2 and H3K27me3 in the PD-L1 expression in TNBC cells. Methods: The change in the expression of PD-L1 at mRNA and protein levels was investigated by establishing an EZH2-knockdown MDA-MB-231 cell line using siRNA followed by RT-qPCR and western blotting analyses. Localization of the PD-L1 protein was assessed using immunofluorescence. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the histone methylation status of PD-L1 promoter regions. The correlation among PD-L1, EZH2, and H3K27me3 protein expressions was explored in 57 patients with TNBC through immunohistochemistry. Results: Knockdown of EZH2 restored the PD-L1 expression and localization of PD-L1 protein in the cellular membrane. ChIP assay revealed that the knockdown of EZH2 diminished H3K27 trimethylation and enhanced H3K4 trimethylation in the promoter region of PD-L1. Immunohistochemical analysis of TNBC specimens reflected an inverse correlation between PD-L1 expression and H3K27me3 nuclear positivity; however, no correlation between H3K27me3 status and EZH2 expression was observed. Conclusions: The downregulation of EZH2 can potentially enhance the efficacy of immune checkpoint inhibitors in patients with TNBC and may provide a new therapeutic strategy.