Tumor-induced DNA methylation in the white blood cells of patients with colorectal cancer

Abstract

The secretions of cancer cells alter epigenetic regulation in cancer stromal cells. The present study investigated the methylation changes in white blood cells (WBCs) caused by the secretions of colorectal cancer (CRC) cells. Changes in the DNA methylation of peripheral blood mononuclear cells (PBMCs) from normal individuals co-cultured with CRC cells were estimated using a methylation microarray. These changes were then compared against the DNA methylation changes and mRNA levels observed in the WBCs of patients with CRC. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) and matrix metalloproteinase 9 (MMP9) were selected to assess the DNA methylation of the WBCs from CRC patients using real-time methylation-specific PCR. The majority of the genes analyzed presented high levels of mRNA in the WBCs of the patients with CRC and DNA methylation in the co-cultured PBMCs. Intragenic methylation revealed the strongest association (P=8.52×10^-21). For validation, MMP9 and PLOD1 were selected and used to test WBCs from 32 patients with CRC and 57 normal controls. The intragenic MMP9 methylation was commonly found (P<0.0001) with high sensitivity (90.63%) and high specificity (96.49%), and a positive predictive value of 93.33% and a negative predictive value of 93.22%. PLOD1 methylation was revealed to have lower sensitivity (30.00%) but higher specificity (97.92%). In addition to circulating WBCs, MMP9 protein expression was observed in infiltrating WBCs and the metastatic lymph nodes of patients with CRC. In conclusion, CRC cells secrete factors that induce genome wide DNA methylation changes in the WBCs of patients with CRC. These changes, including intragenic MMP9 methylation in WBCs, are promising CRC biomarkers to be tested in future CRC screening studies.

Keywords: CRC; WBCs; matrix metalloproteinase 9 methylation.

Figure 1.

A flow chart of the methods employed in the present study. Data were processed and the candidate genes were collected. Group 1 represents DNA methylation changes (GSE110274), and Groups 2 and 3 represent the mRNA levels of the WBCs of patients with CRC (GSE11545 and GSE10715, respectively). All groups were analyzed by connection up- and downregulation expression analysis of microarrays. The association between gene expression and methylation was identified by means of 2×2 contingency tables. Each 2×2 contingency table produced ORs and χ² P-values. The candidate genes were collected from the overlapping genes of the 3 data groups, which were hypermethylated and upregulated genes with a P<0.01 and an OR of >1. Then, the present study selected genes of interest from gene function, biological process and the reported research literatures. The validated genes were quantified methylation using reverse transcription-quantitative methylation-specific PCR in WBCs of patients with CRC compared with normal controls. OR, odds ratio; WBCs, white blood cells; CRC, colorectal cancer; PBMCs, peripheral blood mononuclear cells.

Figure 2.

Methylation in white blood cells as determined by reverse transcription-quantitative methylation-specific PCR. (A) Scatter plot of MMP9 methylation between 32 patients with CRC and 57 normal controls. The levels of MMP9 methylation are expressed as the mean ± standard error of the mean. ***P<0.0001. (B) Scatter plot of PLOD1 methylation between 30 CRC patients and 48 normal controls. The levels of PLOD1 methylation are expressed as the mean ± standard error of the mean. **P=0.0019. (C) ROC curve of MMP9 methylation exhibiting a satisfactory validated gene with high sensitivity (90.63%) and high specificity (96.49%). (D) ROC curve of PLOD1 methylation showing an unsatisfactory validated gene with low sensitivity (30.00%) but high specificity (97.92%). MMP9, matrix metalloproteinase 9; CRC, colorectal cancer; PLOD1, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1; ROC, receiver operator characteristic.

Figure 3.

Formalin-fixed paraffin-embedded samples of patients with CRC were stained with H & E, and immunohistochemistry staining with the anti-MMP9 antibody (magnification, ×200). (A) Normal colon tissue of a patient with CRC exhibited few MMP9 positive WBCs (5–10%). (B) A large number of CRC infiltrating MMP9 positive WBCs (60–70%). (C) Metastatic lymph node of late-stage disease showed a large number of CRC infiltrating MMP9 positive WBCs (50–60%). CRC, colorectal cancer; MMP9, matrix metalloproteinase 9; WBCs, white blood cells; H & E, hematoxylin and eosin.