DNA methylation and repressive H3K9 and H3K27 trimethylation in the promoter regions of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, and PD-L1 genes in human primary breast cancer

Varun Sasidharan Nair, Haytham El Salhat, Rowaida Z. Taha, Anne John, Bassam R. Ali, Eyad Elkord

High expression of immune checkpoints in tumor microenvironment plays significant roles in inhibiting anti-tumor immunity, which is associated with poor prognosis and cancer progression. Major epigenetic modifications in both DNA and histone could be involved in upregulation of immune checkpoints in cancer. Expressions of different immune checkpoint genes and PD-L1 were assessed using qRT-PCR, and the underlying epigenetic modifications including CpG methylation and repressive histone abundance were determined using bisulfite sequencing, and histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) chromatin immunoprecipitation assays (ChIP), respectively. We first assessed the expression level of six immune checkpoints/ligands and found that PD-1, CTLA-4, TIM-3, and LAG-3 were significantly upregulated in breast tumor tissues (TT), compared with breast normal tissues (NT). We investigated the epigenetic modifications beyond this upregulation in immune checkpoint genes. Interestingly, we found that CpG islands in the promoter regions of PD-1, CTLA-4, and TIM-3 were significantly hypomethylated in tumor compared with normal tissues. Additionally, CpG islands of PD-L1 promoter were completely demethylated (100%), LAG-3 were highly hypomethylated (80-90%), and TIGIT were poorly hypomethylated (20-30%), in both NT and TT. These demethylation findings are in accordance with the relative expression data that, out of all these genes, PD-L1 was highly expressed and completely demethylated and TIGIT was poorly expressed and hypermethylated in both NT and TT. Moreover, bindings of H3K9me3 and H3K27me3 were found to be reduced in the promoter loci of PD-1, CTLA-4, TIM-3, and LAG-3 in tumor tissues. Our data demonstrate that both DNA and histone modifications are involved in upregulation of PD-1, CTLA-4, TIM-3, and LAG-3 in breast tumor tissue and these epigenetic modifications could be useful as diagnostic/prognostic biomarkers and/or therapeutic targets in breast cancer.