Aging related methylation influences the gene expression of key control genes in colorectal cancer and adenoma

Abstract

Aim: To analyze colorectal carcinogenesis and age-related DNA methylation alterations of gene sequences associated with epigenetic clock CpG sites.

Methods: In silico DNA methylation analysis of 353 epigenetic clock CpG sites published by Steve Horvath was performed using methylation array data for a set of 123 colonic tissue samples [64 colorectal cancer (CRC), 42 adenoma, 17 normal; GEO accession number: GSE48684]. Among the differentially methylated age-related genes, secreted frizzled related protein 1 (SFRP1) promoter methylation was further investigated in colonic tissue from 8 healthy adults, 19 normal children, 20 adenoma and 8 CRC patients using bisulfite-specific PCR followed by methylation-specific high resolution melting (MS-HRM) analysis. mRNA expression of age-related "epigenetic clock" genes was studied using Affymetrix HGU133 Plus2.0 whole transcriptome data of 153 colonic biopsy samples (49 healthy adult, 49 adenoma, 49 CRC, 6 healthy children) (GEO accession numbers: GSE37364, GSE10714, GSE4183, GSE37267). Whole promoter methylation analysis of genes showing inverse DNA methylation-gene expression data was performed on 30 colonic samples using methyl capture sequencing.

Results: Fifty-seven age-related CpG sites including hypermethylated PPP1R16B, SFRP1, SYNE1 and hypomethylated MGP, PIPOX were differentially methylated between CRC and normal tissues (P < 0.05, Δβ ≥ 10%). In the adenoma vs normal comparison, 70 CpG sites differed significantly, including hypermethylated DKK3, SDC2, SFRP1, SYNE1 and hypomethylated CEMIP, SPATA18 (P < 0.05, Δβ ≥ 10%). In MS-HRM analysis, the SFRP1 promoter region was significantly hypermethylated in CRC (55.0% ± 8.4 %) and adenoma tissue samples (49.9% ± 18.1%) compared to normal adult (5.2% ± 2.7%) and young (2.2% ± 0.7%) colonic tissue (P < 0.0001). DNA methylation of SFRP1 promoter was slightly, but significantly increased in healthy adults compared to normal young samples (P < 0.02). This correlated with significantly increased SFRP1 mRNA levels in children compared to normal adult samples (P < 0.05). In CRC tissue the mRNA expression of 117 age-related genes were changed, while in adenoma samples 102 genes showed differential expression compared with normal colonic tissue (P < 0.05, logFC > 0.5). The change of expression for several genes including SYNE1, CLEC3B, LTBP3 and SFRP1, followed the same pattern in aging and carcinogenesis, though not for all genes (e.g., MGP).

Conclusion: Several age-related DNA methylation alterations can be observed during CRC development and progression affecting the mRNA expression of certain CRC- and adenoma-related key control genes.

Keywords: Adenoma; Aging; Colorectal cancer; DNA methylation; Epigenetic clock; Epigenetic drift; Secreted frizzled related protein 1.

Figure 1

DNA methylation heatmap of normal, adenoma and colorectal carcinoma samples according to the methylation status of age-related CpG sites. From the 353 epigenetic clock CpG sites (cg IDs)[2] significantly differentially methylated in CRC vs normal, adenoma vs normal and CRC vs adenoma comparisons were selected and colonic tissue samples (GSE48684[24]) were classified according to their methylation levels. The analyzed samples are illustrated on X axis, significantly altered CpG sites (cg IDs) are represented on Y axis. DNA methylation intensities (β values) are visualized, red shows hypermethylation, while hypomethylation was marked with green color. CRC: Colorectal carcinoma (light green); Ad: Adenoma (dark blue); N: Normal (light blue).

Figure 2

Genes showing both age- and carcinogenesis-related expression alterations. SYNE1 (spectrin repeat containing nuclear envelope protein 1), LTBP3 (latent transforming growth factor beta binding protein 3) and CLEC3B (C-type lectin domain family 3 member B) genes were downregulated during the colorectal carcinogenesis, similar decreasing expression was found during aging (significantly higher mRNA levels were detected in young colonic samples than in healthy adult biopsy specimens). MGP (matrix Gla protein) was overexpressed in children and in CRC samples compared to adenoma and healthy adults (P < 0.035), hence its opposite expression was found during aging and colorectal carcinogenesis. X axis shows the analyzed sample groups, the normalized mRNA expression can be seen on Y axis. Red dots indicate the normalized mRNA expression values, boxplots represent the medians and standard deviations. Ch: Children; N: Normal; Ad: Adenoma; CRC: Colorectal cancer.

Figure 3

SFRP1 mRNA expression and promoter DNA methylation alterations during aging and in different stages of colorectal carcinogenesis. SFRP1 mRNA expression was significantly downregulated in adenoma and CRC samples compared to normal controls in case of all three Affymetrix probeset IDs representing SFRP1 [202035_s_at: P < 0.05 (A); 202036_s_at: P < 0.0003 (B); 202037_s_at: P < 0.005 (C)]. In colonic biopsy samples of healthy young patients, higher SFRP1 mRNA levels could be measured than in normal adults samples, this overexpression was proven to be significant in two of three transcript IDs [202035_s_at: P < 0.05 (A); 202037_s_at: P < 0.005 (C)]. SFRP1 promoter region was significantly hypermethylated in CRC and adenoma tissue samples compared to normal adult and young colonic tissue (P < 0.0001) (D). In pairwise comparison, DNA methylation of SFRP1 promoter was slightly, but significantly increased in healthy adults compared to normal young samples (P < 0.02) (E). The analyzed sample groups are illustrated on X axis, the normalized mRNA expression (A, B, C) and percentage of SFRP1 promoter methylation (D, E) are represented on Y axis. Red dots indicate the normalized mRNA expression values (A, B, C) and the normalized DNA methylation percentage values (D, E), respectively. Boxplots represent the medians and standard deviations. Ch: Children; N: Normal; Ad: Adenoma; CRC: Colorectal cancer.

Figure 4

SFRP1 protein expression in colonic tissue samples of healthy normal children and adults. Strong/moderate cytoplasmic and nuclear SFRP1 expression both in epithelial and stromal compartments of healthy children (A) and healthy adult (B) samples. Digital microscopic images; magnification × 40; scale: 50 μm.