Gene promoter and exon DNA methylation changes in colon cancer development – mRNA expression and tumor mutation alterations

Colorectal cancer (CRC) is a clinically important malignant disease due to its high incidence and mortality. According to the GLOBOCAN estimates with 1.4 million new cases and 694.000 deaths annually, CRC is the third most common cancer in the world, after lung and breast cancers [1].

The majority of sporadic CRCs develop according to the normal-adenoma-dysplasia-carcinoma sequence described by Fearon and Vogelstein [2]. The accumulation of genetic and epigenetic alterations in colonic epithelium leads to CRC through early and late precancerous adenoma stages in which promoter DNA methylation changes of certain tumor suppressor genes with consecutive mRNA expression changes are one of the earliest events, often prior to the appearance of mutations in well-known genes such as the adenomatosis polyposis coli gene (APC) [3].

Recently, comprehensive molecular characterization of several human cancers including CRC has been performed and the data integrated into The Cancer Genome Atlas (TCGA) database (https://​cancergenome.​nih.​gov/). Integrative evaluation of genetic, epigenetic and gene expression data of hundreds of CRC and paired normal adjacent tissue (NAT) samples revealed that in addition to the known mutations, epigenetic changes (especially DNA methylation) also play a key role in establishing CRC subtypes with different prognostic and therapeutic phenotypes [4]. The majority (84%) of CRCs were found to be non-hypermutated. Non-hypermutated cancers with distinct colonic or rectal location could be distinguished according to copy-number alteration, DNA methylation or gene expression profiles [4].

DNA methylation changes both in promoter and gene body regions contribute to cancer phenotype as they can affect the gene transcription in several ways [3, 5‐7]. In addition to the earlier methods focusing on gene promoter methylation analysis, new technologies, such as BeadChip methylation arrays [4, 8‐12], reduced representation bisulfite sequencing (RRBS) [13], whole genomic bisulfite sequencing (WGBS) and methyl capture sequencing (MethylCap-Seq) [7, 14] were applied to study DNA methylation profiles in CRC. While the majority of investigations included CRC and NAT tissues [4, 9, 10, 12, 14], analysis of precancerous adenomas (AD) are represented in a small number of previous studies [8, 11] including a MethylCap-Seq study of WNT pathway genes we undertook [7]. We have also identified hypermethylated markers (mal, T-cell differentiation protein (MAL), proline rich membrane anchor 1 (PRIMA1), prostaglandin D2 receptor (PTGDR) and secreted frizzled related protein 1 (SFRP1)) in CRC and adenoma using bisulfite sequencing [15] and determined a common ten-gene methylation signature in colorectal adenomas and CRC based on methylation qPCR arrays [16].

BeadChip 27K and 450K arrays and RRBS offer opportunities for analysis of DNA methylation at single nucleotide resolution mainly within CpG islands, however recently developed Epic BeadChip arrays – besides examination of CpG island methylation - allow more extensive study of CpG sites outside of CpG islands, as well. WGBS provides the most widespread whole methylome analysis at single nucleotide resolution, but it is not commonly used due to its high cost. MethylCap-seq is an alternative genome-wide methylation analysis technique to identify novel differentially methylated regions (DMRs) [17, 18]. It gives extensive information about both promoter and gene body methylation, though at lower resolution [18]. Unlike BeadChip arrays, it is suitable for investigation of mutation hot spot regions within the gene body. It is known that mutations can cause altered DNA methylation and DNA methylation changes also can lead to development of mutations [19, 20]. The mutation rate is higher at methylated CpG sites than non-methylated ones [21, 22]. The change of 5-methylcytosine to thymine via spontaneous deamination [23, 24] ‘which is less effectively repaired by the DNA repair machinery than the cytosine to uracil deamination reaction’ [22, 23] can cause the increased mutability of cytosines within CpG sites.

The aim of this study was to analyze genome-wide tissue DNA methylation differences along the colorectal normal-adenoma-carcinoma sequence progression, including gene body methylation changes using MethylCap-seq. The second aim was to search for a potential relation between DNA methylation and mutation alterations for 12 CRC-associated genes. The possible effects of the genetic and epigenetic changes on neoplastic phenotype at transcriptome level were also examined.

The accumulation of DNA methylation alterations accompanied by genetic changes such as mutations and deletions is known to contribute to the pathogenesis of various cancer types including CRC [3, 4]. Comprehensive DNA methylation changes found in precancerous adenoma stages can serve as early detection markers [7, 8, 11, 34]. In this study, global DNA methylation alterations were analyzed along the colorectal normal-adenoma-carcinoma sequence, and top differentially methylated genes/regions were identified using genome-wide MethylCap-seq analysis. The second aim of the study was to find out if there is a potential correlation between DNA methylational and mutational alterations for 12 CRC-associated genes. Furthermore, the possible effects of the genetic and epigenetic changes on TP53 signaling pathway genes at the transcriptome level were also examined.

Global hypomethylation was detected by LINE-1 bisulfite sequencing in CRC samples compared to normal tissue in line with previous data [35‐37]. Although to a lower extent, global DNA hypomethylation could be detected as early as the AD stage. LINE-1 bisulfite sequencing was used for overall hypomethylation analysis due to its superior advance over MethylCap-seq, which predominantly targets genomic regions with high methylated CpGs density [14].

In this study, we identified 22 novel AD- and/or CRC-associated hypermethylated DMRs (approximately one fourth of top50 hypermethylated DMRs) which could be assigned to genes with previously undescribed methylation changes in cancers including CRC. These markers are principally involved in transcription regulation (e.g. BHLHE23, CUX2, HLX, MAFB, MKX, NKX1–1, and GSC2), transport processes (e.g. SLC24A2, GLRA3, LRRC38, SNAP91), and intracellular signaling (e.g. RGS20, GNAL, NRG3). Among the hypermethylated transcription factors, the expression of H2.0 like homeobox (HLX) was found to be reduced in moderately differentiated CRCs [38]. Moreover, HLX is also considered as a tumor suppressor in hepatocellular carcinoma [39]. The platelet derived growth factor receptor alpha (PDGFRA) was observed to be hypermethylated in AD compared to normal controls in our study. It was found to be overexpressed in CRC, but - in accordance with promoter hypermethylation detected in our MethylCap-seq study – it was down-regulated in adenomatous polyps [40]. Nevertheless, one fifth of hypermethylated and the majority of hypomethylated DMRs could not be associated with known genes, both in CRC versus NAT and AD versus NAT comparisons. The identified significant top50 methylation changes could be observed in a high proportion (> 80%) of the specimens within a sample group compared to the mutational alterations analyzed in this study.

On the basis of the methylation levels of the top50 hypomethylated and hypermethylated markers determined in this study, including the newly identified DMRs, the clear separation of CRC and NAT samples was also apparent for an independent sample set (Additional file 2: Figure S2). Furthermore, a partially overlapping set of samples also showed consistent DNA methylation profiles analyzed by MethylCap-seq and EpiTect Methyl qPCR methods (Additional file 2: Figure S3).

Approximately half of the top50 identified hypermethylated DMRs in CRC represent genes found to demonstrate elevated DNA methylation levels in different types of cancers [14, 16, 34, 41‐60]. Seven of the top50 markers (BNC1 [16], DKK2 [48‐50], HS3ST2 [51], MIR124–3 [52], SDC2 [14, 34, 53, 54], TFPI2 [55, 56] and ZIC1 [57]) were previously described as methylated genes in CRC. Hypermethylation of basonuclin (BNC1) zinc finger protein, SDC2 transmembrane heparin sulfate proteoglycan, and DKK2 dickkopf WNT signaling pathway inhibitor 2 genes were also reported in previous studies by our research group [16, 34]. Among the annotated AD versus NAT top50 hypermethylated DMRs, several markers were found to be hypermethylated in various cancers including FLI1 [58, 59], GATA4 [51] and NGFR [60]. These showed elevated methylation levels in CRC samples in other studies.

In this study, we present a comparative analysis between the promoter methylation and mRNA expression data of 12 genes frequently mutated during colorectal carcinogenesis and progression. The results revealed that DNA methylation can play a role in the regulation of APC and CTNNB1 expression in addition to and in parallel with the mutational changes. The above genes are members of the WNT signaling pathway investigated in details in our previous analysis [7]. Hypermethylation of the APC promoter [7, 50, 61, 62] and hypomethylation of the CTNNB1 promoter [7, 49] in AD and CRC samples have also been detected in other studies indicating that the DNA methylation alterations of frequently mutated canonical WNT pathway key genes can contribute to its constitutive activation in colorectal carcinogenesis from the premalignant adenoma stage.

Farkas et al. evaluated DNA methylation changes of genes frequently mutated in CRC using BeadChip450K technology, including 11 of the 12 genes analyzed in our study [49] and reported hypomethylation in CTNNB1 and SMAD2 promoters in CRC compared to NAT samples. Decreased promoter DNA methylation levels of these genes were also observed in our MethylCap-seq analysis together with methylation alterations of other genes such as SMAD4 and TP53 promoters during colorectal carcinogenesis.

In the current project, DNA methylation alterations were also detected in the mutation hot-spot regions of 12 analyzed CRC-associated frequently mutated genes including TP53, APC, KRAS, BRAF, and FBXW7. In accordance with the observation that C - T transitions at CpG sites are the most prevalent mutations in TP53 gene in colon tumors [63], the high mutation rate and methylation changes at mutation hot spot regions of this gene could be detected in our study. DNA methylation can cause mutations in tumor suppressor genes such as TP53, as mutations occur 10–40 times more frequently on the basis of methylated cytosine than of unmethylated cytosine [19, 20]. The conversion of 5-methylcytosine to thymine via spontaneous deamination [23, 24] or by the APOBEC/AID system [64] can lead to a high mutational burden of 5-methylcytosine. The 5-methylcytosine can be involved in increased mutability through other mechanisms. According to a recent report, elevated C to G transversion rate in cancer genomes can be associated with 5-hydroxymethylcytosines derived by the oxidation of 5-methylcytosine catalyzed by TET proteins [65].

Hypomethylation was also detected in addition to the elevated methylation levels on certain mutation hot-spots. This is only seemingly contradictory to previous data indicating that the mutation rate is higher on methylated CpG sites than on unmethylated ones [21], as the relative hypomethylation (from high level to intermediate level) and not the absolute loss of DNA methylation was observed on certain mutation hot-spots in our study. It is in conjunction with the results of a recent work describing that among the methylated CpG sites, the rate of mutations (or SNP density) was found to be increased on less methylated CpG sites (20–60%) as compared to high-intermediately and highly methylated CpGs (60–80%; > 80%) [21, 66]. Cancer-associated overall hypomethylation of the genome including heterochromatic DNA repeats, retrotransposons, and endogenous retroviral elements also contribute to genome instability [20].

In our analysis, DMRs could be identified on all chromosomes with the relatively largest number of aligned sequence reads on chromosome 17, similar to the MethylCap-seq study performed by Simmer et al. [14]. Next, DNA methylation alterations of TP53 (encoded on chr 17) signaling pathway genes were also investigated. TP53 pathway deregulation frequently occurs through the mutations or deletion of TP53 itself [67]. Outside the mutations of the TP53 gene, this pathway is rarely hit by any other mutations/polymorphisms [68‐70]. Other mechanisms, such as epigenetic regulation including DNA methylation changes of TP53 pathway genes, also contribute to attenuating the pathway and participate in cancer development [67], and TP53 itself is also thought to regulate cancer-associated genes showing altered methylation patterns [71]. Accordingly, our MethylCap-Seq analysis revealed significant promoter DNA methylation changes in approximately one third of TP53 signaling pathway genes in CRC. Moreover, an even greater proportion of TP53 pathway gene promoters (around 40%) showed altered DNA methylation in AD samples compared to NAT controls. The alterations of the identified TP53 pathway genes with inverse promoter DNA methylation and mRNA expression differences (Table 2) were found to be associated with tumorigenesis in different cancer types including CRC [72‐88]. Among these markers, in addition to the down-regulation of well known p16 (CDKN2A) [75, 76] and p21 (CDKN1A) [74] cyclin dependent kinase inhibitors, BCL2 associated X, apoptosis regulator (BAX) [72], SESN2 [85‐87], IGFBP3 [84] and cytochrome c, somatic (CYCS) [77] are also thought to exert tumor suppressor functions. Diminished or loss of CYCS protein expression in AD and CRC tissue was found to be correlated with apoptosis resistance [77]. DDB2 damage specific DNA binding protein, which was described to suppress the tumorigenicity in case of ovarian cancer [78] and reduces CRC invasiveness [79], showed promoter hypomethylation and overexpression in AD samples in our study, suggesting its contribution to the inhibition of uncontrolled expansion in the adenoma stage.

Using genome-wide DNA methylation analysis, we identified novel aberrant methylation profiles of genes including HLX, CUX2, MKX, NRG3 and PDGFRA associated with the colorectal adenoma-carcinoma sequence progression. In addition to the genetic changes, DNA methylation alterations were also shown in the mutation hot-spot regions of 12 analyzed, CRC-associated, frequently mutated genes including, TP53, APC, KRAS, BRAF, and FBXW7. Global hypomethylation – which might be linked to genetic instability - could be detected as early as the adenoma stage.

Our study also revealed that promoter DNA methylation changes influence the mRNA expression level in the case of a significant part of the TP53 pathway genes. Thus epigenetic alterations can also contribute to a whole pathway-related effect on DNA repair and apoptosis in addition to single gene (e.g. TP53) mutations.

In summary, the methyl-capture sequencing technique yielded reproducible, clinically relevant results on the whole genome level which are related to cancer phenotype development through mRNA expression changes and to the cancer genotype through the link of mutation formation.