DNA methylome in visceral adipose tissue can discriminate patients with and without colorectal cancer

Abstract

Adipose tissue dysfunction, particularly the visceral (VAT) compartment, has been proposed to play a relevant role in colorectal cancer (CRC) development and progression. Epigenetic mechanisms could be involved in this association. The current study aimed to evaluate if specific epigenetic marks in VAT are associated with colorectal cancer (CRC) to identify epigenetic hallmarks of adipose tissue-related CRC. Epigenome-wide DNA methylation was evaluated in VAT from 25 healthy participants and 29 CRC patients, using the Infinium HumanMethylation450K BeadChip. The epigenome-wide methylation analysis identified 170,184 sites able to perfectly separate the CRC and healthy samples. The differentially methylated CpG sites (DMCpGs) showed a global trend for increased methylated levels in CRC with respect to healthy group. Most of the genes encoded by the DMCpGs belonged to metabolic pathways and cell cycle, insulin resistance, and adipocytokine signalling, as well as tumoural transformation processes. In gene-specific analyses, involved genes biologically relevant for the development of CRC include PTPRN2, MAD1L1, TNXB, DIP2C, INPP5A, HDCA4, PRDM16, RPTOR, ATP11A, TBCD, PABPC3, and IER2. The methylation level of some of them showed a discriminatory capacity for detecting CRC higher than 90%, showing IER2 to have the highest capacity. This study reveals that a specific methylation pattern of VAT is associated with CRC. Some of the epigenetic marks identified could provide useful tools for the prediction and personalized treatment of CRC connected to excess adiposity.

Keywords: DNA methylation; adipose tissue; cancer; microarray; obesity.

Figure 1.

Profile of DNA methylation in visceral adipose tissue from colorectal cancer patients compared with healthy participants. (a) Principal component analysis for DNA methylation levels of 1,000 most variable CpGs between colorectal cancer and tumour free visceral adipose tissue (VAT) samples. (b) . Manhattan plot showing epigenome-wide p-values of association. The y axis shows the – log10(p) values of 455,267 valid CpGs, and the x axis shows their chromosomal position. The horizontal discontinuous line represents the threshold of p< 0.05 for selecting differentially methylated CpG sites. (c). Volcano plot of differences in DNA methylation between colorectal cancer and tumour free VAT samples. Each point represents a CpG site (n = 455,267) with mean differences (fold change) in DNA methylation between groups on the x-axis and -log10 of the corrected p value on the y-axis. Negative methylation differences indicate hypomethylation and positive differences show hypermethylation in the CRC patients compared to the healthy participants. (d) Supervised clustering of the 1,000 DMCpGs that were found to be differentially methylated between CRC patients and healthy subjects (e) Global and (f) promoter region differences in methylation levels between both groups. Asterisk indicates differences statistically significant according to the Wilcoxon test (p< 0.001). Abbreviations: PC, principal component, DMCpG, differentially methylated CpGs.

Figure 2.

Characterization of the visceral adipose tissue-related colorectal cancer DMCpGs. (a) Genomic distribution of the differentially methylated CpG (DMCpGs) and their respective locations regarding the broader CpG context, (b) gene region and (c) chromosome. (d) List of the top of 30 genes mostly enriched by significant CpGs. Grey intermittent line represents a cut-off point of 150 DMCpGs with lower methylation in the CRC than healthy group. (e) Genes represented by filtered criteria, which DMCpGs are in promoter and islands, follow a false discovery rate (FDR) lower than 0.001 and a difference in β-values at least of 15% in absolute values. Abbreviations, DMCpG, differentially methylated CpGs, CGI, CpG island, TSS, transcription start site, UTR, untranslated region, IGR, intergenic region, FDR, false discovery rate, FC, fold change.

Figure 3.

(A) Biological implications of the visceral adipose tissue-related CRC DMCpGs.(a) Summary of the GO analysis of the biological process categories representing the differentially methylated genes located at the promoter region. (b) KEGG pathway analysis of DMCpGs located at the promoter region. (c) Gene–protein interaction network-STRING analysis. Most of the genes regulated by methylation belonged to a network significantly enriched in protein interactions (p < 0.001) according to STRING analysis. Abbreviations: DMCpGs, differentially methylated CpGs; GO, gene ontology, KEGG, Kyoto Encyclopaedia of Genes and Genomes.

Figure 4.

Receiver operating characteristic (ROC) curves for the methylation levels of the CRC-related differentially methylated CpGs in visceral adipose tissue. (a) Differentially methylated CpG site (DMCpGs) from PTPRN2. (b) DMCpGs from MAD1L1. (c) DMCpGs from INPP5A. (d) DMCpGs from HDAC4. (e) DMCpGs from ATP11A. (f) DMCpGs from PRDM16. (g) DMCpGs from TBCD. (h) DMCpGs from PABPC3. (i) DMCpGs from IER2. Abbreviations: AUC, area under the ROC curve.