Cancer epigenetics

Cancer epigenetics is the study of epigenetic modifications to the genome of cancer cells that do not involve a change in the nucleotide sequence. Epigenetic alterations are as important as genetic mutations in a cell’s transformation to cancer. Mechanisms of epigenetic silencing of tumor suppressor genes and activation of oncogenes include: alteration in CpG island methylation patterns, histone modifications, and dysregulation of DNA binding proteins. Understanding epigenetic mechanisms holds great promise for cancer prevention, detection, and therapy.

DNA methylation

A DNA molecule fragment that is methylated at two cytosines

DNA methylation is probably the most well researched epigenetic mark that differs between normal cells and tumor cells in humans. The "normal" CpG methylation profile is often inverted in cells that become tumorigenic[1]. In normal cells, CpG Islands preceding gene promoters are generally unmethylated, 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.

Hypermethylation of gene promoters can result in silencing of those genes. This type of epigenetic mutation is dangerous when genes that regulate the cell cycle are silenced, allowing cells to grow and reproduce uncontrollably, leading to tumorigenesis[1]. Genes commonly found to be transcriptionally silenced due to promoter hypermethylation include: [[Cyclin-dependent kinase inhibitor p16, a cell-cycle inhibitor; p53, a tumor suppressor gene; MGMT, a DNA repair gene; APC, a cell cycle regulator; MLH1, a DNA-repair gene; and BRCA1, another DNA-repair gene[1][19].

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[2-5]. 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 [21]. In healthy cells, CpG dinucleotides of lower densities are found within coding and non-coding intergenic regions. Parasitic repetitive sequences, centromeres and oncogenes are often repressed through methylation.

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[2-5]. 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. [2-5]

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. [19]

Histone modifications

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. Addtionally, 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. Therefore, one might expect the histone modification profiles of healthy and cancerous cells to differ.

In comparison to healthy cells, cancerous cells have been seen to exhibit decreased monoacetylated and trimethylated forms of histone H4 (decreased H4ac and H4me3)[10]. In mouse models, scientists have noticed that the loss of histone H4 acetylation and trimethylation actually increases as tumor growth continues[10]. Interestingly, 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[1, 11].

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 (H3K9me) 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).[8, 9]

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[18]. 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.[18]

Mutations in the epigenetic machinery itself may occur as well, potentially responsible for the changing epigenetic profiles of cancerous cells. For example, the decrease in H4K16ac may be caused by either a decrease in activity of a histone acetyltransferases (HATs) or an increase in deacetylation by SIRT1.[1] 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[12]. These findings indicate a promising mechanism for altering epigenetic profiles through enzymatic inhibition or enhancement.

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.[28]

MicroRNA gene silencing

In mammals, microRNA (miRNA) regulates around 60% of the transcriptional activity of protein-encoding genes. Some miRNAs have also been found to undergo methylation-associated silencing in cancerous cells[6, 7]. Let-7 and miR15/16 play important roles in down-regulating RAS and BCL2 oncogenes, and their silencing has been found in cancer cells[21]. A decrease in 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 indicate a negative effect of methylation on the function and expression of miR-125b1, therefore Soto-Reyes and his team were able to conclude that DNA methylation has a part in silencing the gene. Furthermore, results show that some miRNA’s are epigenetically silenced early on in breast cancer, and therefore these miRNA’s could potentially be useful as tumor markers. [27]

Cancer subtype specific epigenetic modifications

Prostate cancer

Prostate cancer kills around 35,000 men yearly, and about 220,000 men are diagnosed with prostate cancer per year, in North America alone.[29] 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.[29] Prostate cancer has been associated with gene silencing by CpG island hypermethylation. The GSTP1 gene has been found to defend prostate cells against genomic damage that is caused by different oxidants or carcinogens.[30] This suggests that the silencing of this gene will permit genetic damage to the prostate by oxidants and carcinogens. Methylation ofcytosines within a CpG island is a causative factor in gene silencing. Modern epigenetics have correlated CpG island hypermethylation with unexpressed genes. Hypermethylation of the CpG island within the promoter region of the GSTP1 gene has been found to occur in more than 90% of prostate cancers.[30] The high percentage of occurrence demonstrates that hypermethylation of the CpG island within the promoter region of the GSTP1 gene is a highly causative factor of prostate carcinogenesis. In prostate cancer, many other genes have been found to be hypermethylated. An experiment using quantitative real-time methylation-specific polymerase chain reaction (PCR) viewed 16 different genes of prostate cancer that were hypermethylated in high frequencies of the CpG islands in GSTP1, APC, RASSF1a, PTGS2, and MDRI, but in normal prostate tissues almost no methylation was found.[30] Polymerase chain reaction shows the fragment sizes of DNA of interest. Hypermethylation of the genes were shown to be unexpressed. This demonstrates that the hypermethylation of the 16 different genes show no expression of these genes in prostate cancer, and that these genes are necessary for a normal functioning disease-free prostate.

Cervical cancer

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).[31] Furthermore, cervix intraepithelial neoplasia (CIN) is primarily caused by oncogenic HPV16.[31] 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.[31] Only 2/16 CpG sites tested in HPV16 upstream regulatory region were found to have association with increased methylation in CIN3+.[31] 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.[31] Furthermore, 1/3 of CpG sites in mitochondrial DNA were associated with increased methylation in CIN3+.[31] Thus, a correlation exists between CIN3+ and increased methylation of CpG sites in the HPV16 L1 open reading frame.[31] This could be a potential biomarker for future screens of cancerous and precancerous cervical disease.[31]

Current therapeutics

5-aza-2’-deoxycytidine

Drugs that specifically target the inverted methylation pattern of cancerous cells include the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine. This hypomethylating agent has been used to treat myelodysplastic syndrome, a blood cancer produced by abnormal bone marrow stem cells[19]. An inhibitor for all three types of active DNA methyltransferases, 5-azaC, previously thought to be highly toxic for human trials, proves to be an effective therapeutics in clinical trials when apply in low dosage, reducing progression of myelodisplastic syndrome to leukaemia, and increasing survival rate of patients with cancer [22].

Histone deacetylase (HDAC) inhibitors have undergone many clinical trials. With significant efficacy in treating T cell lymphoma, two HDAC inhibitors, vorinostat and romidepsin, have recently been approved by the Food and Drug Administration[23][24]. However, since these HDAC inhibitors alter the acetylation state of many proteins in addition to the histone of interest, knowledge of the underlying mechanism at the molecular level of patient response is required to enhance the efficiency of using such inhibitors as treatment[21]. A histone deacetylase, suberoylanilide hydroxamic acid (SAHA) has had some success in clinical trials [20]. Epigenetic therapeutics are taken in combination and concurrently with traditional cancer treatments, such as chemotherapy and immunotherapy. There is much crosstalk between CpG island methylation and histone modifications. Treatment with HDAC inhibitors has been found to promote gene reactivation after DNA methyl-transferases inhibitors have repressed transcription[25].

Tools for identification

Previously, epigenetic profiles were limited to individual genes under scrutiny by a particular research team. Recently, however, scientists have been moving toward a more genomic approach to determine an entire genomic profile for cancerous versus healthy cells.[1]

Popular approaches for measuring CpG methylation in cells include:

• Bisulfite sequencing
• Combined bisulfite restriction analysis (COBRA)
• MethyLight
• Pyrosequencing
• Restriction landmark genomic scanning
• Arbitrary primed PCR
• HELP assay (HpaII tiny fragment enrichment by ligation-mediated PCR)
• Chromatin immunoprecipitation ChIP-Chip using antibodies specific for methyl-CpG binding domain proteins
• Methylated DNA immunoprecipitation Methyl-DIP
• Gene-expression profiles via DNA microarray : comparing mRNA levels from cancer cell lines before and after treatment with a demethylating agent

Since bisulfite sequencing is considered the gold standard for measuring CpG methylation, when one of the other methods is used, results are usually confirmed using bisulfite sequencing[1]. Popular approaches for determining histone modification profiles in cancerous versus healthy cells include[1]:

• Mass spectrometry
• Chromatin Immunoprecipitation Assay

Diagnostic and prognostic uses

Researchers are hoping to identify specific epigenetic profiles of various types and subtypes of cancer with the goal of using these profiles as tools to diagnose individuals more specifically and accurately[1]. Since epigenetic profiles change, scientists would like to use the different epigenomic profiles to determine the stage of development or level of aggressiveness of a particular cancer in patients. For example, hypermethylation of the genes coding for Death-Associated Protein Kinase (DAPK), p16, and Epithelial Membrane Protein 3 (EMP3) have been linked to more aggressive forms of lung, colorectal, and brain cancers[5]. This type of knowledge can affect the way that doctors will diagnose and choose to treat their patients.

Another factor that will influence the treatment of patients is knowing how well they will respond to certain treatments. Personalized epigenomic profiles of cancerous cells can provide insight into this field. For example, MGMT is an enzyme that reverses the addition of alkyl groups to the nucleotide guanine[13]. Alkylating guanine, however, is the mechanism by which several chemotherapeutic drugs act in order to disrupt DNA and cause cell death[14-17]. Therefore, if the gene encoding MGMT in cancer cells is hypermethylated and in effect silenced or repressed, the chemotherapeutic drugs that act by methylating guanine will be more effective than in cancer cells that have a functional MGMT enzyme.

Epigenetic biomarkers can also be utilized as tools for molecular prognosis. In primary tumor and mediastinal lymph node biopsy samples, hypermethylation of both CDKN2A and CDH13 serves as the marker for increased risk of faster cancer relapse and higher death rate of patients [26].