The absence of B7-H4 inhibits PD-L1 expression by driving a methylation of PD-L1 promoter in breast cancer cells.

The expression of B7-H4, a co-inhibitory molecule highly detected in cancer cells, serves as an important prognostic marker and therapeutic target in breast cancer. B7-H4 has been shown to modulate the malignancy of breast cancer cells through affecting cell stemness and the epithelial-mesenchymal transition (EMT). However, whether B7-H4 influences the expression of other immunoregulatory molecules, and how this crosstalk contributes to the clinicopathological features of breast cancer remain controversial. Although the majority of the tumors are positive for B7-H4 and negative for PD-L1, we noticed a portion of breast tumor cells (4.3–18.8%) showing co-expression of two molecules. Besides, there is an overall positive correlation in the expression of B7-H4 and PD-L1 in breast cancer. Mechanistically, B7-H4 promotes the transcription of PD-L1 via downregulating the DNA methyltransferase 1 (DNMT1)-mediated methylation in cg19724470 and cg15837913 CpG loci in the promoter of PD-L1. Moreover, the expression of B7-H4 is negatively associated with the methylation levels of these two loci in a breast cancer cohort in the TCGA database, and is necessary for interferon-γ (IFN-γ)-induced PD-L1 expression in breast cancer cells. Pharmacological inhibition of DNMT1 improves IFN-γ responsiveness in breast cancer cells. Functionally, individuals with co-expression of B7-H4 and PD-L1 present higher abundance of CD8A, granzyme B (GZMB) and perforin 1 (PRF1) than those without co-expression in tumor tissues, indicating increased T cell infiltration in these patients. Consistently, the co-expression of these two molecules is also associated with improved disease-free survival in breast cancer patients. Overall, our study unveils a mechanistic link between B7-H4 and PD-L1 expression through DNA methylation-mediated epigenetic regulation in breast cancer, and indicates that epigenetic therapy holds potential in remodeling the immune landscape and overcoming treatment resistance in breast cancer.