All mammalian cells descended from a fertilized egg (a zygote) share a common DNA sequence (except for new mutations in some lineages). However, during development and formation of different tissues epigenetic factors change. The changes include histone modifications, CpG island methylations and chromatin reorganizations which can cause the stable silencing or activation of particular genes. Once differentiated tissues are formed, CpG island methylation is generally stably inherited from one cell division to the next through the DNA methylation maintenance machinery.
In cancer, a number of mutational changes are found in protein coding genes. Colorectal cancers typically have 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations that silence protein expression in the genes affected. However, transcriptional silencing may be more important than mutation in causing gene silencing in progression to cancer. In colorectal cancers about 600 to 800 genes are transcriptionally silenced, compared to adjacent normal-appearing tissues, by CpG island methylation. Transcriptional repression in cancer can also occur by other epigenetic mechanisms, such as altered expression of microRNAs.
CpG islands are frequent control elements
CpG islands are commonly 200 to 2000 base pairs long, have a C:G base pair content >50%, and have frequent 5' → 3' CpG sequences. About 70% of human promoters located near the transcription start site of a gene contain a CpG island.
Promoters located at a distance from the transcription start site of a gene also frequently contain CpG islands. The promoter of the DNA repair gene ERCC1, for instance, was identified and located about 5,400 nucleotides upstream of its coding region. CpG islands also occur frequently in promoters for functional noncoding RNAs such as microRNAs and Long non-coding RNAs (lncRNAs).
Methylation of CpG islands in promoters stably silences genes
Genes can be silenced by multiple methylation of CpG sites in the CpG islands of their promoters.
Even if silencing of a gene is initiated by another mechanism, this
often is followed by methylation of CpG sites in the promoter CpG island
to stabilize the silencing of the gene. On the other hand, hypomethylation of CpG islands in promoters can result in gene over-expression.
Promoter CpG hyper/hypo-methylation in cancer
In
cancers, loss of expression of genes occurs about 10 times more
frequently by hypermethylation of promoter CpG islands than by
mutations. For instance, in colon tumors compared to adjacent
normal-appearing colonic mucosa, about 600 to 800 heavily methylated CpG
islands occur in promoters of genes in the tumors while these CpG
islands are not methylated in the adjacent mucosa. In contrast, as Vogelstein et al. point out, in a colorectal cancer there are typically only about 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations.
DNA repair gene silencing in cancer
In
sporadic cancers, a DNA repair deficiency is occasionally found to be
due to a mutation in a DNA repair gene. However, much more frequently,
reduced or absent expression of a DNA repair gene in cancer is due to
methylation of its promoter. For example, of 113 colorectal cancers
examined, only four had a missense mutation in the DNA repair gene MGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region. Similarly, among 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene PMS2 expression, 6 had a mutation in the PMS2
gene, while for 103 PMS2 was deficient because its pairing partner MLH1
was repressed due to promoter methylation (PMS2 protein is unstable in
the absence of MLH1).
In the remaining 10 cases, loss of PMS2 expression was likely due to
epigenetic overexpression of the microRNA, miR-155, which down-regulates
MLH1.
Frequency of hypermethylation of DNA repair genes in cancer
Twenty-two
DNA repair genes with hypermethylated promoters, and reduced or absent
expression, were found to occur among 17 types of cancer, as listed in
two review articles. As listed in one of the reviews, promoter hypermethylation of MGMT
occurs frequently in a number of cancers including 93% of bladder
cancers, 88% of stomach cancers, 74% of thyroid cancers, 40%-90% of
colorectal cancers and 50% of brain cancers. That review also indicated
promoter hypermethylation of LIG4, NEIL1, ATM, MLH1 or FANCB occurs at frequencies between 33% to 82% in one or more of head and neck cancers, non-small-cell lung cancers or non-small-cell lung cancer squamous cell carcinomas. The article Werner syndrome ATP-dependent helicase indicates the DNA repair gene WRN has a promoter that is frequently hypermethylated in a number of cancers, with hypermethylation occurring in 11% to 38% of colorectal, head and neck, stomach, prostate, breast, thyroid, non-Hodgkin lymphoma, chondrosarcoma and osteosarcoma cancers.
Likely role of hypermethylation of DNA repair genes in cancer
As discussed by Jin and Roberston in their review,
silencing of a DNA repair gene by hypermethylation may be a very early
step in progression to cancer. Such silencing is proposed to act
similarly to a germ-line mutation in a DNA repair gene, and predisposes
the cell and its descendants to progression to cancer. Another review
also indicated an early role for hypermethylation of DNA repair genes
in cancer. If a gene necessary for DNA repair is hypermethylated,
resulting in deficient DNA repair, DNA damages will accumulate.
Increased DNA damage tends to cause increased errors during DNA
synthesis, leading to mutations that can give rise to cancer.
If hypermethylation of a DNA repair gene is an early step in
carcinogenesis, then it may also occur in the normal-appearing tissues
surrounding the cancer from which the cancer arose (the field defect). See the table below.
| Cancer | Gene | Frequency in Cancer | Frequency in Field Defect |
|---|---|---|---|
| Colorectal | MGMT | 55% | 54% |
| Colorectal | MSH2 | 13% | 5% |
| Colorectal | WRN | 29% | 13% |
| Head and Neck | MGMT | 54% | 38% |
| Head and Neck | MLH1 | 33% | 25% |
| Non-small cell lung cancer | ATM | 69% | 59% |
| Non-small cell lung cancer | MLH1 | 69% | 72% |
| Stomach | MGMT | 88% | 78% |
| Stomach | MLH1 | 73% | 20% |
| Esophagus | MLH1 | 77%-100% | 23%-79% |
While DNA damages may give rise to mutations through error prone translesion synthesis, DNA damages can also give rise to epigenetic alterations during faulty DNA repair processes.
The DNA damages that accumulate due to hypermethylation of the
promoters of DNA repair genes can be a source of the increased
epigenetic alterations found in many genes in cancers.
In an early study, looking at a limited set of transcriptional promoters, Fernandez et al.
examined the DNA methylation profiles of 855 primary tumors. Comparing
each tumor type with its corresponding normal tissue, 729 CpG island
sites (55% of the 1322 CpG island sites evaluated) showed differential
DNA methylation. Of these sites, 496 were hypermethylated (repressed)
and 233 were hypomethylated (activated). Thus, there is a high level of
promoter methylation alterations in tumors. Some of these alterations
may contribute to cancer progression.
DNA methylation of microRNAs in cancer
In mammals, microRNAs (miRNAs) regulate the transcriptional activity of about 60% of protein-encoding genes.
Individual miRNAs can each target, and repress transcription of, on
average, roughly 200 messenger RNAs of protein coding genes. The promoters of about one third of the 167 miRNAs evaluated by Vrba et al.
in normal breast tissues were differentially hyper/hypo-methylated in
breast cancers. A more recent study pointed out that the 167 miRNAs
evaluated by Vrba et al. were only 10% of the miRNAs found expressed in
breast tissues. This later study found that 58% of the miRNAs in breast tissue had differentially methylated regions in their promoters in breast cancers, including 278 hypermethylated miRNAs and 802 hypomethylated miRNAs.
One miRNA that is over-expressed about 100-fold in breast cancers is miR-182. MiR-182 targets the BRCA1 messenger RNA and may be a major cause of reduced BRCA1 protein expression in many breast cancers.
microRNAs that control DNA methyltransferase genes in cancer
Some miRNAs target the messenger RNAs for DNA methyltransferase genes DNMT1, DNMT3A and DNMT3B, whose gene products are needed for initiating and stabilizing promoter methylations. As summarized in three reviews,
miRNAs miR-29a, miR-29b and miR-29c target DNMT3A and DNMT3B; miR-148a
and miR-148b target DNMT3B; and miR-152 and miR-301 target DNMT1. In
addition, miR-34b targets DNMT1 and the promoter of miR-34b itself is
hypermethylated and under-expressed in the majority of prostate cancers.
When expression of these microRNAs is altered, they may also be a
source of the hyper/hypo-methylation of the promoters of protein-coding
genes in cancers.
