Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content

Abstract

Background Hypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules’ (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect. Methods LINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules’ in situ level (n = 40). Results Significantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes—which met our analysing criteria—showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05). Conclusion Our results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

CTpathway: a CrossTalk-based pathway enrichment analysis method for cancer research

“…To evaluate reproducibility, we used both microarray data from the GEO database [38] and RNA-seq data from the TCGA database [39] for each of four cancer types (COAD, LIHC, LUAD, and OV). We downloaded eight gene expression data of four cancer types from the GEO database (GSE100179 [40], GSE101685 [41], GSE116959 [42], GSE9891 [43]) and TCGA database (Additional file 1: Table S2). Each data set includes case (cancer) and control (normal) samples (details in Additional file 1: Table S2).…”

“…We also identified pathways unique to a single cancer. For example, transport-related pathways ("Transferrin endocytosis and recycling" and "Passive transport by Aquaporins") were reproducibly identified in COAD in both the GSE100179 [40] and TCGA patient cohort and not in other cancers. Many metabolism-linked pathways ("Pyruvate metabolism, " "Glycerolipid metabolism, " "Glycogen degradation II, " "Acetate conversion to acetyl-CoA, " and "Caffeine metabolism") were specifically identified in LIHC patient cohorts in TCGA and GEO (GSE101685 [41]).…”

“…nih. gov/ geo/) under accession numbers GSE100179 [40], GSE101685 [41], [42], and GSE9891 [43] and the TCGA database [39] (https:// portal. gdc.…”

Methodological and Biological Factors Influencing Global DNA Methylation Results Measured by LINE-1 Pyrosequencing Assay in Colorectal Tissue and Liquid Biopsy Samples

“…Global DNA hypomethylation is a molecular driving force in cancer development through mobile genetic elements – such as LINE-1 retrotransposons - reactivation [ 1 , 2 , 41 ]; hence, LINE-1 methylation level possesses important information as a prognostic and diagnostic value in cancer diseases, including colorectal cancer [ 8 , 9 , 10 , 42 ]. In this study, our aim was to comprehensively examine the potential methodological, technical, and biological factors that can influence the LINE-1 methylation measurements in a colon tissue, buffy coat, and plasma sample cohort.…”

“…Global DNA hypomethylation is an early molecular event in colorectal cancer (CRC) leading to genetic instability through the reactivation of mobile genetic elements, especially of the long interspersed nuclear element 1 (LINE-1) sequences [ 1 , 2 ]. Global DNA and LINE-1 methylation levels have a prognostic value [ 3 , 4 , 5 , 6 , 7 ]; moreover, LINE-1 methylation also possesses diagnostic potential in a cancer screening procedure analyzed in plasma samples obtained from patients with colorectal tumors [ 8 , 9 , 10 ]. Hence, it is essential to measure LINE-1 methylation levels in experimental and clinical fields in a reliable, comparable, and repeatable way.…”