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. 2018 Nov 10;8(2):e1542918.
doi: 10.1080/2162402X.2018.1542918. eCollection 2019.

DNA methylation of immune checkpoints in the peripheral blood of breast and colorectal cancer patients

Asma A Elashi  1 , Varun Sasidharan Nair  1 , Rowaida Z Taha  1 , Hibah Shaath  1 , Eyad Elkord  1   2
Affiliations

DNA methylation of immune checkpoints in the peripheral blood of breast and colorectal cancer patients

Asma A Elashi et al. Oncoimmunology. .

Abstract

Aberrant expression of immune checkpoints (ICs) in cancer creates an immunosuppressive microenvironment, which supports immune evasion of tumor cells. We have recently reported that epigenetic modifications are critical for ICs expression in the tumor microenvironment (TME) of primary breast cancer (PBC) and colorectal cancer (CRC). Herein, we investigated transcriptomic expression of ICs (PD-1, CTLA-4, LAG-3, TIM-3, TIGIT) and PD-L1 in peripheral blood of PBC and CRC patients, compared to healthy donors (HD). We found that expressions of TIM-3, TIGIT, PD-L1 were significantly upregulated, while LAG-3 expression was downregulated in peripheral blood of PBC and CRC patients. Demethylation enzymes TET2 and TET3 were also upregulated. In addition, promoter DNA methylation status of PD-1 was significantly hypermethylated, while PD-L1 was hypomethylated in PBC and CRC patients. Furthermore, TIGIT was significantly hypomethylated only in CRC patients. Remarkably, promoter methylation status of LAG-3, TIGIT and PD-L1 was in concordance with transcriptomic expression in CRC: the more the hypomethylation, the higher the expression. In comparison, we found that CTLA-4, TIM-3, TIGIT and PD-L1 in PBC, and CTLA-4 in CRC patients were significantly upregulated in peripheral blood, compared with tumor tissues of the same patients. However, demethylation status of all ICs was higher in TT, except for TIGIT in PBC, and CTLA-4 in CRC patients. These data indicate that the underlying mechanisms behind peripheral upregulation of PD-L1 and TIGIT in cancer patients could be due to aberrant promoter methylation profile. Moreover, demethylation inhibitors together with anti-PD-L1/anti-TIGIT could be a more efficient therapeutic strategy in cancer patients.

Keywords: Breast cancer; Colorectal cancer; DNA methylation; immune checkpoints.

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Figures

Figure 1.
Figure 1.
Relative expression of immune checkpoints in circulation of HD, PBC and CRC patients. RNA from PBC, CRC and HD patients were isolated and reversely transcribed into cDNA, followed by quantitative RT-PCR to assess the relative expression levels of PD-1 (a), CTLA-4 (b), TIM-3 (c), LAG-3 (d), TIGIT (e), PD-L1 (f) and VISTA (g) in circulation. All genes were normalized to β-actin.
Figure 2.
Figure 2.
Relative expression of ICs/ligand in PBC and CRC patients based on their TNM staging and histological grade. Patients were classified according to their TNM and histological garde. Scatter plots of PBC (a) and CRC (c) patients of TNM stage I and II (1) compared to stage III and IV (2), and PBC (b) and CRC (d) patients of histological grade moderate and well differentiated (a) were compared to poorly differentiated (b).
Figure 3.
Figure 3.
Relative expression of methylation/demethylation enzymes in circulation of HD, PBC and CRC patients. Scatter plots show the relative expression of DNMT3a (a), DNMT3b (b), TET2 (c), and TET3 (d) in circulation of PBC, CRC and HD patients. All genes were normalized to β-actin.
Figure 4.
Figure 4.
Analysis of CpG methylation status of ICs and PD-L1 in circulation of HD, PBC and CRC patients. Representative plots show the promoter CpG methylation status in PD-1 (a), CTLA-4 (b), TIM-3 (c), LAG-3 (d), TIGIT (e) and PD-L1 (f) in the circulation of 10 HD, 8 PBC and 12 CRC patients as analyzed by bisulfite sequencing of the gDNA. Methylation status of individual CpG motifs is shown by white (demethylation) or grey (methylation) colors.
Figure 5.
Figure 5.
Overall CpG demethylation percentage of ICs and PD-L1 in HD, PBC and CRC patients. Bar plots show the average demethylation percentage of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and PD-L1 in 10 HD, 8 PBC and 12 CRC patients.
Figure 6.
Figure 6.
Comparisons of the relative expression and DNA demethylation percentage of the ICs/PD-L1 in blood, normal and tumor tissues from PBC and CRC patients. Bar plots show the relative expression of ICs/PD-L1 (a) and CRC (c) patients. Bar plots show the CpG demethylation status of the ICs/PD-L1 in blood, NT and TT of PBC (b) and CRC (d) patients.
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