Epigenetic changes (aberrant DNA methylation) in colorectal neoplasia

Abstract

Both genetic and epigenetic events have been implicated in the stepwise histological progression involving adenoma-carcinoma and hyperplastic polyp/serrated adenoma-carcinoma sequences in the development of colorectal cancer. Genetic changes have been observed at each step in the initiation and progression of polyps to adenocarcinomas. Epigenetic changes also occur at each step in the pathogenesis of colorectal cancers and include CpG island DNA hypermethylation in the promoter region of genes resulting in transcriptional silencing through associated changes in chromatin structure and effects on binding of transcription factors, and DNA global hypomethylation which leads to chromosomal instability. Recent studies on MLH1 and APC genes indicate that epigenetic and genetic changes cooperate to facilitate tumor initiation and progression. Since aberrant CGI DNA promoter hypermethylation can be detected not only in colorectal polyps and cancers, but also in sera and stool, hypermethylated genes may serve as molecular markers for early detection, risk assessment and diagnosis. In addition, silenced genes caused by CGI DNA promoter hypermethylation can be reactivated by demethylating agents and also by both the inhibitors of DNA methyltransferases and histone deacetylases. Therefore, these epigenetically acting drugs should be evaluated for their chemopreventive and therapeutic potential for colorectal cancers.

Keywords: Colorectal cancer; Colorectal polyps; CpG island; DNA hypomethylation; DNA methylation; Epigenetic changes.

Fig. 1

Overall scheme of key genetic and epigenetic events in colorectal tumorigenesis. Both genetic and epigenetic changes play important roles in colorectal tumorigenesis. Genetic changes may be broadly classified into two categories; chromosomal instability (CIN) and microsatellite instability (MSI). Although about 80% of tumors exhibit chromosomal instability, stability of microsatellite DNA and involves chromosomal aberrations such as loss of heterozygosity. The typical genetic events associated with tumors in this pathway are shown. MSI is found in most cases of hereditary non-polyposis colorectal cancer and in 15% of sporadic tumors. This pathway involves inactivation of DNA mismatch repair genes caused by promoter hypermethylation of hMLH1 followed by mutations in the microsatellite sequences of the genes important in tumor progression such as TGFβ RII and BAX. MSI is a late event in colorectal tumorigenesis and chromosomal aberrations are rare in MSI tumors. There are two major epigenetic aberrations that occur during colorectal tumorigenesis, global DNA hypomethylation and region specific or promoter hypermethylation of CpG islands (CGI). Global DNA hypomethylation occurs early in colorectal tumorigenesis (aberrant crypt foci/hyperplastic polyps) and remains at constant levels during tumor progression. It is frequently associated with chromosomal instability. CGI promoter hypermethylation also occurs early in colorectal tumorigenesis, but the propensity of methylation increases with tumor progression. In addition, different frequencies and patterns of promoter methylation of specific genes are observed during tumor initiation and progression. CGI promoter hypermethylation is frequently associated with BRAF mutation and MSI.

Fig. 2

Common DNA methylation changes observed in cancer. In normal tissues, the majority of CpG islands in the promoters of tumor suppressor genes and genes regulating cell cycle and apoptosis are methylation-free and are expressed normally. However, repetitive sequences and interspersed CpG dinucleotides are heavily methylated. The genome of cancer cells is characterized by regional or promoter hypermethylation of CpG islands and/or reduction in the number of methylated CpG dinucleotides in the repetitive sequences and interspersed regions, so-called global DNA hypomethylation. In cancer cells, 3 types of combination of these two events may occur. Cancer A with promoter hypermethylation and global DNA methylation, Cancer B with unmethylated promoters with global DNA hypomethylation and Cancer C with promoter hypermethylation and global DNA hypomethylation. Cancer A and cancer B occur more frequently than cancer C.

Fig. 3

Genetic and epigenetic changes that inactivate tumor suppressor genes according to Knudson's two-hit hypothesis. When caused only by genetic changes, the first hit may be somatic mutation within the coding region of one copy of the gene. The second hit generally involves loss of the chromosomal region of the other copy of the gene or loss of heterozygosity. The loss of both alleles of a tumor suppressor gene leads to a series of events that causes malignant transformation. Aberrant promoter hypermethylation can have the same effect as a coding region mutation in one copy of the gene (the first hit) and is frequently associated with the loss of other copy of the gene by either somatic loss of the chromosomal region or by aberrant promoter hypermethylation of the other copy (biallelic methylation) which represents the second hit.

Fig. 4

CpG island methylation status in the promoter and the related chromatin structures in transcriptional activation and silencing. In normal cells, the CpG sites adjacent to transcription start site in the promoter of the genes are frequently unmethylated. The transcriptional machinery is activated by the binding of transcriptional factors (TFs), co-acting factors (CAs) and histone acetyltransferases (HATs) in this region. Thus, the gene promoter shown on the upper left is transcriptionally active. Upstream and downstream of this region, CpG islands are methylated by DNA methyltransferases (DNMTs). In these regions methylcytosine-binding proteins (MBPs) that bind to methylated CpG sites recruit histone deacetylases (HDACs) and histone methyltransferases to form a complex. Stop signs (barriers) indicate the mechanisms involved in preventing the spreading of CpG island methylation to unmethylated transcriptionally active region of the promoter. Left bottom shows the related chromatin structure around the transcriptionally active, unmethylated promoter occupied by nucleosomes composed of histone complexes. The lysine residues in the tails of histone H3 are acetylated (acK). Lysine 4 is methylated (mK4) and lysine 9 is unmethylated (K9). These changes contribute to open and relaxed conformation of the chromatin allowing key components of the gene transcription apparatus accessible to this region of the promoter. In the regions, upstream and downstream of transcriptionally active promoter regions, the lysine residues are deacetylated (K), and methylated (mK9) respectively and the chromatin structures have closed and dense conformation. In cancer cells depicted in the upper right, the barriers for the spreading of CpG island methylation are removed. Methylation spreads toward the promoter region near the transcription start site resulting in transcriptional silencing. Thus, the gene promoter shown on the upper right is transcriptionally inactive. These events result in closed and dense chromatin conformation making it difficult for the key components of gene transcription apparatus to bind to the promoter contributing further to transcriptional repression.

Fig. 5

Wnt signaling pathway is regulated by SFRPs, epigenetic gatekeepers and APC; a genetic gatekeeper. In normal colon epithelial cells, secreted frizzled related proteins (SFRPs) inhibit Wnt signaling by competing with Wnt ligand for binding to their receptor, Frizzled (FRZ). In cancer cells, however, methylation of CpG islands of the promoters of SFRPs inhibits their transcription causing the Wnt signaling pathway to become active. Thus, SFRPs function as an epigenetic gatekeeper. When Wnt signaling is inactive in normal colon epithelial cells, the APC complex phosphorylates β-catenin leading to its degradation preventing the nuclear accumulation of β-catenin and its binding to Lef/Tcf HMG box transcription factors. These events result in the differentiation and homeostasis of colonic epithelial cells. In cancer cells, mutated APC allow unphosphorylated β-catenin to translocate to nucleus and activate transcription of genes that promote cell proliferation and survival. Therefore, APC expression functions as a genetic gatekeeper.