Cancer epigenetics

A DNA molecule fragment that is methylated at two cytosines

In somatic cells, patterns of DNA methylation are in general transmitted to daughter cells with high fidelity.[8] Typically, this methylation only occurs at cytosines that are located 5' to guanosine in the CpG dinucleotides of higher order eukaryotes.[9] However, epigenetic DNA methylation differs between normal cells and tumor cells in humans. The "normal" CpG methylation profile is often inverted in cells that become tumorigenic.[10] In normal cells, CpG islands preceding gene promoters are generally unmethylated, and tend to be transcriptionally active, while other individual CpG dinucleotides throughout the genome tend to be methylated. However, in cancer cells, CpG islands preceding tumor suppressor gene promoters are often hypermethylated, while CpG methylation of oncogene promoter regions and parasitic repeat sequences is often decreased.[11]

Hypermethylation of tumor suppressor gene promoter regions can result in silencing of those genes. This type of epigenetic mutation allows cells to grow and reproduce uncontrollably, leading to tumorigenesis.[10] The addition of methyl groups to cytosines causes the DNA to coil tightly around the histone proteins, resulting in DNA that can not undergo transcription (transcriptionally silenced DNA). Genes commonly found to be transcriptionally silenced due to promoter hypermethylation include: Cyclin-dependent kinase inhibitor p16, a cell-cycle inhibitor; MGMT, a DNA repair gene; APC, a cell cycle regulator; MLH1, a DNA-repair gene; and BRCA1, another DNA-repair gene.[10][12] Indeed, cancer cells can become addicted to the transcriptional silencing, due to promoter hypermethylation, of some key tumor suppressor genes, a process known as epigenetic addiction.[13]

Hypomethylation of CpG dinucleotides in other parts of the genome leads to chromosome instability due to mechanisms such as loss of imprinting and reactivation of transposable elements.[14][15][16][17] Loss of imprinting of insulin-like growth factor gene (IGF2) increases risk of colorectal cancer and is associated with Beckwith-Wiedemann syndrome which significantly increases the risk of cancer for newborns.[18] In healthy cells, CpG dinucleotides of lower densities are found within coding and non-coding intergenic regions. Expression of some repetitive sequences and meiotic recombination at centromeres are repressed through methylation [19]

The entire genome of a cancerous cell contains significantly less methylcytosine than the genome of a healthy cell. In fact, cancer cell genomes have 20-50% less methylation at individual CpG dinucleotides across the genome.[14][15][16][17] CpG islands found in promoter regions are usually protected from DNA methylation. In cancer cells CpG islands are hypomethylated [20] The regions flanking CpG islands called CpG island shores are where most DNA methylation occurs in the CpG dinucleotide context. Cancer cells are deferentially methylated at CpG island shores. In cancer cells, hypermethylation in the CpG island shores move into CpG islands, or hypomethylation of CpG islands move into CpG island shores eliminating sharp epigenetic boundaries between these genetic elements.[21] In cancer cells "global hypomethylation" due to disruption in DNA methyltransferases (DNMTs) may promote mitotic recombination and chromosome rearrangement, ultimately resulting in aneuploidy when the chromosomes fail to separate properly during mitosis.[14][15][16][17]

CpG island methylation is important in regulation of gene expression, yet cytosine methylation can lead directly to destabilizing genetic mutations and a precancerous cellular state. Methylated cytosines make hydrolysis of the amine group and spontaneous conversion to thymine more favorable. They can cause aberrant recruitment of chromatin proteins. Cytosine methylations change the amount of UV light absorption of the nucleotide base, creating pyrimidine dimers. When mutation results in loss of heterozygosity at tumor suppressor gene sites, these genes may become inactive. Single base pair mutations during replication can also have detrimental effects.[12]

Eukaryotic DNA has a complex structure. It is generally wrapped around special proteins called histones to form a structure called a nucleosome. A nucleosome consists of 2 sets of 4 histones: H2A, H2B, H3, and H4. Additionally, histone H1 contributes to DNA packaging outside of the nucleosome. Certain histone modifying enzymes can add or remove functional groups to the histones, and these modifications influence the level of transcription of the genes wrapped around those histones and the level of DNA replication. Histone modification profiles of healthy and cancerous cells tend to differ.

In comparison to healthy cells, cancerous cells exhibit decreased monoacetylated and trimethylated forms of histone H4 (decreased H4ac and H4me3).[22] Additionally, mouse models have shown that a decrease in histone H4R3 asymmetric dimethylation (H4R3me2a) of the p19ARF promoter is correlated with more advanced cases of tumorigenesis and metastasis.[23] In mouse models, the loss of histone H4 acetylation and trimethylation increases as tumor growth continues.[22] Loss of histone H4 Lysine 16 acetylation (H4K16ac), which is a mark of aging at the telomeres, specifically loses its acetylation. Some scientists hope this particular loss of histone acetylation might be battled with a histone deacetylase (HDAC) inhibitor specific for SIRT1, an HDAC specific for H4K16.[10][24]

Other histone marks associated with tumorigenesis include increased deacetylation (decreased acetylation) of histones H3 and H4, decreased trimethylation of histone H3 Lysine 4 (H3K4me3), and increased monomethylation of histone H3 Lysine 9 (H3K9me1) and trimethylation of histone H3 Lysine 27 (H3K27me3). These histone modifications can silence tumor suppressor genes despite the drop in methylation of the gene's CpG island (an event that normally activates genes).[25][26]

Some research has focused on blocking the action of BRD4 on acetylated histones, which has been shown to increase the expression of the Myc protein, implicated in several cancers. The development process of the drug to bind to BRD4 is noteworthy for the collaborative, open approach the team is taking.[27]

The tumor suppressor gene p53 regulates DNA repair and can induce apoptosis in dysregulated cells. E Soto-Reyes and F Recillas-Targa elucidated the importance of the CTCF protein in regulating p53 expression.[28] CTCF, or CCCTC binding factor, is a zinc finger protein that insulates the p53 promoter from accumulating repressive histone marks. In certain types of cancer cells, the CTCF protein does not bind normally, and the p53 promoter accumulates repressive histone marks, causing p53 expression to decrease.[28]

Mutations in the epigenetic machinery itself may occur as well, potentially responsible for the changing epigenetic profiles of cancerous cells. The histone variants of the H2A family are highly conserved in mammals, playing critical roles in regulating many nuclear processes by altering chromatin structure. One of the key H2A variants, H2A.X, marks DNA damage, facilitating the recruitment of DNA repair proteins to restore genomic integrity. Another variant, H2A.Z, plays an important role in both gene activation and repression. A high level of H2A.Z expression is detected in many cancers and is significantly associated with cellular proliferation and genomic instability.[11] Histone variant macroH2A1 is important in the pathogenesis of many types of cancers, for instance in hepatocellular carcinoma.[29] Other mechanisms include a decrease in H4K16ac may be caused by either a decrease in activity of a histone acetyltransferases (HATs) or an increase in deacetylation by SIRT1.[10] Likewise, an inactivating frameshift mutation in HDAC2, a histone deacetylase that acts on many histone-tail lysines, has been associated with cancers showing altered histone acetylation patterns.[30] These findings indicate a promising mechanism for altering epigenetic profiles through enzymatic inhibition or enhancement. A new emerging field that captures toxicological epigenetic changes as a result of the exposure to different compounds (drugs, food, and environment) is toxicoepigenetics. In this field, there is growing interest in mapping changes in histone modifications and their possible consequences.[31]

DNA damage, caused by UV light, ionizing radiation, environmental toxins, and metabolic chemicals, can also lead to genomic instability and cancer. The DNA damage response to double strand DNA breaks (DSB) is mediated in part by histone modifications. At a DSB, MRE11-RAD50-NBS1 (MRN) protein complex recruits ataxia telangiectasia mutated (ATM) kinase which phosphorylates Serine 129 of Histone 2A. MDC1, mediator of DNA damage checkpoint 1, binds to the phosphopeptide, and phosphorylation of H2AX may spread by a positive feedback loop of MRN-ATM recruitment and phosphorylation. TIP60 acetylates the γH2AX, which is then polyubiquitylated. RAP80, a subunit of the DNA repair breast cancer type 1 susceptibility protein complex (BRCA1-A), binds ubiquitin attached to histones. BRCA1-A activity arrests the cell cycle at the G2/M checkpoint, allowing time for DNA repair, or apoptosis may be initiated.[32]

In mammals, microRNAs (miRNAs) regulate about 60% of the transcriptional activity of protein-encoding genes.[33] Some miRNAs also undergo methylation-associated silencing in cancer cells.[34][35] Let-7 and miR15/16 play important roles in down-regulating RAS and BCL2 oncogenes, and their silencing occurs in cancer cells.[18] Decreased expression of miR-125b1, a miRNA that functions as a tumor suppressor, was observed in prostate, ovarian, breast and glial cell cancers. In vitro experiments have shown that miR-125b1 targets two genes, HER2/neu and ESR1, that are linked to breast cancer. DNA methylation, specifically hypermethylation, is one of the main ways that the miR-125b1 is epigenetically silenced. In patients with breast cancer, hypermethylation of CpG islands located proximal to the transcription start site was observed. Loss of CTCF binding and an increase in repressive histone marks, H3K9me3 and H3K27me3, correlates with DNA methylation and miR-125b1 silencing. Mechanistically, CTCF may function as a boundary element to stop the spread of DNA methylation. Results from experiments conducted by Soto-Reyes et al.[36] indicate a negative effect of methylation on the function and expression of miR-125b1. Therefore, they concluded that DNA methylation has a part in silencing the gene. Furthermore, some miRNA's are epigenetically silenced early on in breast cancer, and therefore these miRNA's could potentially be useful as tumor markers.[36] The epigenetic silencing of miRNA genes by aberrant DNA methylation is a frequent event in cancer cells; almost one third of miRNA promoters active in normal mammary cells were found hypermethylated in breast cancer cells - that is a several fold greater proportion than is usually observed for protein coding genes.[37]

Deficiencies in DNA repair proteins that function in accurate DNA repair pathways increase the risk of mutation. Mutation rates are strongly increased in cells with mutations in DNA mismatch repair[73][74] or in homologous recombinational repair (HRR).[75] Individuals with inherited mutations in any of 34 DNA repair genes are at increased risk of cancer (see DNA repair defects and increased cancer risk).

In sporadic cancers, a deficiency in DNA repair is occasionally found to be due to a mutation in a DNA repair gene, but much more frequently reduced or absent expression of DNA repair genes is due to epigenetic alterations that reduce or silence gene expression. For example, for 113 colorectal cancers examined in sequence, 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 (an epigenetic alteration).[76] Similarly, out of 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene PMS2 expression, PMS2 protein was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1).[48] In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1.[47]

Epigenetic defects in DNA repair genes are frequent in cancers. In the table, multiple cancers were evaluated for reduced or absent expression of the DNA repair gene of interest, and the frequency shown is the frequency with which the cancers had an epigenetic deficiency of gene expression. Such epigenetic deficiencies likely arise early in carcinogenesis, since they are also frequently found (though at somewhat lower frequency) in the field defect surrounding the cancer from which the cancer likely arose (see Table).

It appears that cancers may frequently be initiated by an epigenetic reduction in expression of one or more DNA repair enzymes. Reduced DNA repair likely allows accumulation of DNA damages. Error prone translesion synthesis past some of these DNA damages may give rise to a mutation with a selective advantage. A clonal patch with a selective advantage may grow and out-compete neighboring cells, forming a field defect. While there is no obvious selective advantage for a cell to have reduced DNA repair, the epimutation of the DNA repair gene may be carried along as a passenger when the cells with the selectively advantageous mutation are replicated. In the cells carrying both the epimutation of the DNA repair gene and the mutation with the selective advantage, further DNA damages will accumulate, and these could, in turn, give rise to further mutations with still greater selective advantages. Epigenetic defects in DNA repair may thus contribute to the characteristic high frequency of mutations in the genomes of cancers, and cause their carcinogenic progression.

Cancers have high levels of genome instability, associated with a high frequency of mutations. A high frequency of genomic mutations increases the likelihood of particular mutations occurring that activate oncogenes and inactivate tumor suppressor genes, leading to carcinogenesis. On the basis of whole genome sequencing, cancers are found to have thousands to hundreds of thousands of mutations in their whole genomes.[87] (Also see Mutation frequencies in cancers.) By comparison, the mutation frequency in the whole genome between generations for humans (parent to child) is about 70 new mutations per generation.[88][89] In the protein coding regions of the genome, there are only about 0.35 mutations between parent/child generations (less than one mutated protein per generation).[90] Whole genome sequencing in blood cells for a pair of identical twin 100-year-old centenarians only found 8 somatic differences, though somatic variation occurring in less than 20% of blood cells would be undetected.[91]

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.[41][42][92][93] The DNA damages that accumulate due to epigenetic DNA repair defects 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.[94] 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 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 epigenetic promoter methylation alterations in tumors. Some of these epigenetic alterations may contribute to cancer progression.

Melanoma is a deadly skin cancer that originates from melanocytes. Several epigenetic alterations are known to play a role in the transition of melanocytes to melanoma cells. This includes DNA methylation that can be inherited without making changes to the DNA sequence, as well as silencing the tumor suppressor genes in the epidermis that have been exposed to UV radiation for periods of time.[102] The silencing of tumor suppressor genes leads to photocarcinogenesis which is associated to epigenetic alterations in DNA methylation, DNA methyltransferases, and histone acetylation.[102] These alterations are the consequence of deregulation of their corresponding enzymes. Several histone methyltransferases and demethylases are among these enzymes.[103]

Prostate cancer kills around 35,000 men yearly, and about 220,000 men are diagnosed with prostate cancer per year, in North America alone.[104] Prostate cancer is the second leading cause of cancer-caused fatalities in men, and within a man's lifetime, one in six men will have the disease.[104] Alterations in histone acetylation and DNA methylation occur in various genes influencing prostate cancer, and have been seen in genes involved in hormonal response.[105] More than 90% of prostate cancers show gene silencing by CpG island hypermethylation of the GSTP1 gene promoter, which protects prostate cells from genomic damage that is caused by different oxidants or carcinogens.[106] Real-time methylation-specific polymerase chain reaction (PCR) suggests that many other genes are also hypermethylated.[106] Gene expression in the prostate may be modulated by nutrition and lifestyle changes.[107]

The second most common malignant tumor in women is invasive cervical cancer (ICC) and more than 50% of all invasive cervical cancer (ICC) is caused by oncongenic human papillomavirus 16 (HPV16).[108] Furthermore, cervix intraepithelial neoplasia (CIN) is primarily caused by oncogenic HPV16.[108] As in many cases, the causative factor for cancer does not always take a direct route from infection to the development of cancer. Genomic methylation patterns have been associated with invasive cervical cancer. Within the HPV16L1 region, 14 tested CpG sites have significantly higher methylation in CIN3+ than in HPV16 genomes of women without CIN3.[108] Only 2/16 CpG sites tested in HPV16 upstream regulatory region were found to have association with increased methylation in CIN3+.[108] This suggests that the direct route from infection to cancer is sometimes detoured to a precancerous state in cervix intraepithelial neoplasia. Additionally, increased CpG site methylation was found in low levels in most of the five host nuclear genes studied, including 5/5 TERT, 1/4 DAPK1, 2/5 RARB, MAL, and CADM1.[108] Furthermore, 1/3 of CpG sites in mitochondrial DNA were associated with increased methylation in CIN3+.[108] Thus, a correlation exists between CIN3+ and increased methylation of CpG sites in the HPV16 L1 open reading frame.[108] This could be a potential biomarker for future screens of cancerous and precancerous cervical disease.[108]

Recent studies have shown that the mixed-lineage leukemia (MLL) gene causes leukemia by rearranging and fusing with other genes in different chromosomes, which is a process under epigenetic control.[109] Mutations in MLL block the correct regulatory regions in leukemia associated translocations or insertions causing malignant transformation controlled by HOX genes.[110] This is what leads to the increase in white blood cells. Leukemia related genes are managed by the same pathways that control epigenetics, signaling transduction, transcriptional regulation, and energy metabolism. It was indicated that infections, electromagnetic fields and increased birth weight can contribute to being the causes of leukemia.[111]

There are about 15,000 new cases of sarcoma in the US each year, and about 6,200 people were projected to die of sarcoma in the US in 2014.[112] Sarcomas comprise a large number of rare, histogenetically heterogeneous mesenchymal tumors that, for example, include chondrosarcoma, Ewing's sarcoma, leiomyosarcoma, liposarcoma, osteosarcoma, synovial sarcoma, and (alveolar and embryonal) rhabdomyosarcoma. Several oncogenes and tumor suppressor genes are epigenetically altered in sarcomas. These include APC, CDKN1A, CDKN2A, CDKN2B, Ezrin, FGFR1, GADD45A, MGMT, STK3, STK4, PTEN, RASSF1A, WIF1, as well as several miRNAs.[113] Expression of epigenetic modifiers such as that of the BMI1 component of the PRC1 complex is deregulated in chondrosarcoma, Ewing's sarcoma, and osteosarcoma, and expression of the EZH2 component of the PRC2 complex is altered in Ewing's sarcoma and rhabdomyosarcoma. Similarly, expression of another epigenetic modifier, the LSD1 histone demethylase, is increased in chondrosarcoma, Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma. Drug targeting and inhibition of EZH2 in Ewing's sarcoma,[114] or of LSD1 in several sarcomas,[115] inhibits tumor cell growth in these sarcomas.

Lung cancer is the second most common type of cancer and leading cause of death in men and women in the United States, it is estimated that there is about 216,000 new cases and 160,000 deaths due to lung cancer.[116]

Initiation and progression of lung carcinoma is the result of the interaction between genetic, epigenetic and environmental factors. Most cases of lung cancer are because of genetic mutations in EGFR, KRAS, STK11 (also known as LKB1), TP53 (also known as p53), and CDKN2A (also known as p16 or INK4a)[117][118][119] with the most common type of lung cancer being an inactivation at p16. p16 is a tumor suppressor protein that occurs in mostly in humans the functional significance of the mutations was tested on many other species including mice, cats, dogs, monkeys and cows the identification of these multiple nonoverlapping clones was not entirely surprising since the reduced stringency hybridization of a zoo blot with the same probe also revealed 10-15 positive EcoRI fragments in all species tested.[120]