DNA methylation and repressive histones in the promoters of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, PD-L1, and galectin-9 genes in human colorectal cancer

Varun Sasidharan Nair, Salman M Toor, Rowaida Z Taha, Hibah Shaath, Eyad Elkord, Varun Sasidharan Nair, Salman M Toor, Rowaida Z Taha, Hibah Shaath, Eyad Elkord

Abstract

Background: Colorectal cancer (CRC) is the third most commonly diagnosed human malignancy worldwide. Upregulation of inhibitory immune checkpoints by tumor-infiltrating immune cells (TIICs) or their ligands by tumor cells leads to tumor evasion from host immunosurveillance. Changes in DNA methylation pattern and enrichment of methylated histone marks in the promoter regions could be major contributors to the upregulation of immune checkpoints (ICs) in the tumor microenvironment (TME).

Methods: Relative expressions of various immune checkpoints and ligands in colon normal tissues (NT) and colorectal tumor tissues (TT) were assessed by qRT-PCR. The epigenetic modifications behind this upregulation were determined by investigating the CpG methylation status of their promoter regions using bisulfite sequencing. Distributions of histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) in promoter regions of these genes were assessed by chromatin immunoprecipitation (ChIP) assay.

Results: We found that the expression levels of PD-1, CTLA-4, TIM-3, TIGIT, PD-L1, and galectin-9 were significantly higher in colorectal tumor tissues, compared with colon normal tissues. To study the role of DNA methylation, we checked the promoter CpG methylation of ICs and ligands and found that only CTLA-4 and TIGIT, among other genes, were significantly hypomethylated in TT compared with NT. Next, we checked the abundance of repressive histones (H3K9me3 and H3K27me3) in the promoter regions of ICs/ligands. We found that bindings of H3K9me3 in PD-1 and TIGIT promoters and H3K27me3 in CTLA-4 promotor were significantly lower in TT compared with NT. Additionally, bindings of both H3K9me3 and H3K27me3 in the TIM-3 promoter were significantly lower in TT compared with NT.

Conclusion: This study shows that both DNA hypomethylation and H3K9me3 and H3K27me3 repressive histones are involved in upregulation of CTLA-4 and TIGIT genes. However, repressive histones, but not DNA hypomethylation, are involved in upregulation of PD-1 and TIM-3 genes in CRC tumor tissue. These epigenetic modifications could be utilized as diagnostic biomarkers for CRC.

Keywords: Colorectal cancer; DNA methylation; Galectin-9; Histone trimethylation; Immune checkpoints; PD-L1.

Conflict of interest statement

Ethics approval and consent to participate

The study was executed under ethical approval by Qatar Biomedical Research Institute, Doha, Qatar (Protocol No. 2017-006). All patients provided written informed consent prior to sample collection.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Expression of immune checkpoints/ligands and methylation/demethylation genes in colorectal tumor and normal colon tissues. RNA isolated from tissues from 14 patients was reverse transcribed to cDNA. Quantitative RT-PCR was performed to assess the expression level of immune checkpoints PD-1, CTLA-4, TIM-3, LAG-3, and TIGIT (a); immune checkpoint ligands PD-L1 and galectin-9 (b); demethylation/methylation enzymes TET1, TET2, TET3, DNMT3a, and DNMT3b (c) from both NT and TT. The relative expression of each gene was normalized to β-actin
Fig. 2
Fig. 2
Analyses of CpG methylation of immune checkpoint promoters in colorectal tumor and normal tissues. Representative plots show the CpG methylation of the promoter regions together with bar charts of the demethylation percentages of PD-1 (a), CTLA-4 (b), TIM-3 (c), LAG-3 (d), TIGIT (e), PD-L1 (f), and galectin-9 (g) as analyzed by bisulfite sequencing of the genomic DNA isolated from colorectal tumor and normal colon tissues from 14 patients. Methylation status of individual CpG motifs is shown by white (demethylation) or gray (methylation) colors
Fig. 3
Fig. 3
Corrected demethylation percentage of immune checkpoint promoters in tumor tissues. CpG methylation status of the promoter regions of PD-1, CTLA-4, TIM-3, LAG-3, PD-L1, TIGIT, and galectin-9 was analyzed by bisulfite sequencing of the genomic DNA isolated from colorectal tumor and normal colon tissues from 14 patients. A bar diagram shows the average demethylation percentage from the 14 NT and TT samples of each gene (a). A bar diagram shows the corrected demethylation percentage of immune checkpoints by subtracting average demethylation percentage of NT from TT (b). A bar diagram shows the corrected demethylation percentage of immune checkpoints (c) and their ligands (d) in 14 individual patients
Fig. 4
Fig. 4
Analyses of distribution of H3K9me3 and H3K27me3 in the promoters of immune checkpoints/ligands in colorectal tumor and normal colon tissues. Cells from five individual NT and TT samples were isolated by enzyme disaggregation. Chromatin was precipitated using anti-H3 as control, anti-H3K9me3, anti-H3K27me3 antibodies, and IgG as negative control. Subsequent qPCR was performed using promoter primers for PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, PD-L1, and galectin-9. Data were normalized to input. ChIP analysis of distribution of H3, H3K9me3, and H3K27me3 at PD-1 (a), CTLA-4 (b), TIM-3 (c), LAG-3 (d), TIGIT (e), PD-L1 (f), and galectin-9 (g) promoters are shown

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