We have a dedicated site for Germany. This volume discusses certain epigenetic changes recognized in early carcinogenic lesions and different tumors, as well as factors that alter the epigenome and epigenetic profile such as diet, alcohol, immunity, circadian rhythm, and more. The chapters in this book further delve into this field and cover topics such as epigenome-based precision medicine in lung cancer; interplay between genetic and epigenetic changes in breast cancer subtypes; genetic regulation of PDCD1 gene in cancer immunology; and pyrosequencing methylation analysis.
Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and authoritative, Cancer Epigenetics for Precision Medicine: Methods and Protocols is a valuable resource to help researchers and scientists identify these specific biomarkers and work towards the prevention, diagnosis, and prognosis of different cancers in the future.
DNA methyltransferases and histone deacetylases are the two major drug targets for epigenetic inhibition to date, although others are expected to be added in the near future Table 1. Most of the drugs with promising clinical prospects or those used widely as research tools are included in Table 1. Among those not listed are analogs of the methyl group donor S -adenosylmethionine, which inhibit a broad spectrum of cellular methyltransferases and have been shown to be potentially mutagenic Although most reports on the inhibitors listed in Table 1 assume a high drug target specificity, many of these drugs in fact have pleiotropic effects.
Moreover, 5-aza-CdR has been shown to be capable of transcriptionally activating genes with unmethylated promoters 48 , also leading to increased acetylation and H3 lysine 4 methylation 37 , suggesting this drug can induce chromatin remodeling independently of its effects on cytosine methylation. Gene expression microarray experiments confirmed that many genes activated by 5-aza-CdR lack promoter methylation, and for reasons that are not entirely clear, appear to be enriched for genes involved in interferon signaling 22 — 24 , 49 — Similar studies have now also been performed using zebularine The network of multiple reinforcing interactions involved in epigenetic silencing suggests that combination therapy would be a particularly appropriate strategy to achieve clinical efficacy 42 — Indeed, combinations of DNA methyltransferase and histone deacetylase inhibitors appear to synergize effectively in the reactivation of epigenetically silenced genes 25 , 36 , 38 , 53 , Combination trials are underway to test this concept in the clinic.
Several other exciting developments in epigenetic therapy have emerged recently. First, insight into the mode of action, the mechanism of toxicity and the pharmacokinetics of decitabine inspired an improved protocol with prolonged administration at lower doses, with equal, if not better efficacy than previous regimens Toxicity has been one of the major problems with this drug and the efficacy of such low-dose protocols is an important advance. Even more exciting is the recent addition of zebularine to the arsenal of DNA methyltransferase inhibitors 56 , In contrast to decitabine, zebularine is stable in aqueous solution and can be administered orally, greatly simplifying continuous low-dose therapy 56 , Zebularine appears to be more effectively incorporated by cancer cells than by fibroblasts Such a preferential response of cancer cells to epigenetic therapy is shared by decitabine 51 and histone deacetylase inhibitors 59 , also in non-proliferating cancer cells, suggesting that epigenetic therapy could be an effective strategy for treating tumors with a low mitotic index Epigenetic changes in cancer cells not only provide novel targets for drug therapy but also offer unique prospects for cancer diagnostics The three main approaches to assess the epigenetic state of individual gene loci are to 1 measure gene expression, 2 determine histone modifications and chromatin protein composition and 3 analyze promoter DNA methylation status.
Chromatin immunoprecipitation has been an extremely useful research tool to analyze chromatin protein composition and modifications. However, it has not advanced sufficiently yet to become a clinically useful diagnostic method, in contrast to serum proteomics by mass spectrometry, which is progressing rapidly in clinical feasibility studies Gene expression microarray analysis has proved to be a powerful method for identifying novel subclasses of cancer and predicting clinical outcome or response to therapy 62 , However, gene expression analysis is generally not viewed as epigenetic analysis, in part because mechanistic understanding of gene regulation evolved from studies of transcriptional control by transcription factors, which does not necessarily involve mitotically stable epigenetic change, although the fields of gene regulation and epigenetics are moving closer.
The major interest in cancer epigenetics as a diagnostic tool is in localized epigenetic silencing.
The use of gene expression microarray studies to identify non-transcribed genes as candidates for promoter CpG island hypermethylation has had limited success, because lack of gene expression can stem from other causes aside from epigenetic silencing 22 — 24 , 49 — For the most part, cancer epigenetics has relied on measurements of CpG island DNA hypermethylation 7 , DNA methylation markers are used in cancer diagnostics for both disease classification and disease detection.
As a classification tool, CpG island hypermethylation is generally analyzed on sufficient quantities of primary tissue such as a surgically resected tumor sample.
The DNA methylation status of individual gene promoters can be used for general prognosis or to predict response to a particular therapy. There have been numerous reports describing an association between hypermethylation of individual genes and overall clinical outcome prognosis for various types of cancer 64 — Individual methylation markers have also been linked to breast cancer metastasis In particular, methylation of the E-cadherin CDH1 promoter appears to be required for invasion and metastasis 72 — It is more difficult to make a convincing case that a DNA methylation marker is a predictor of response to a specific therapy, and not just a general prognostic marker of clinical outcome, independent of therapy.
One of the best cases has been made for hypermethylation of the O 6 -methylguanine methyltransferase MGMT promoter, which is associated with increased survival in glioma patients treated with alkylating agents 76 — Melanoma cells with acquired resistance to the antineoplastic alkylating compound fotemustine, by repeated in vitro drug exposure, were shown to have reactivated the MGMT gene Increasingly, the profiling of a broader set of DNA methylation markers is used for prognosis and prediction, often facilitated by hierarchical cluster analysis A screen of 10 genes in neuroblastoma samples was able to delineate three main clinical risk groups 81 , whereas a panel of 35 methylation markers revealed an association of DNA methylation profiles with hormone receptor status and response to tamoxifen treatment in breast cancer patients Unsupervised clustering of unselected CpG islands in 19 late-stage ovarian tumors allowed discrimination between two major subgroups differing in progression-free survival rates The increasing use of DNA methylation microarrays, high-throughput bisulfite genomic sequencing, mass spectrometry and other genome-wide techniques such as restriction landmark genome scanning is rapidly genomicizing epigenetics 25 , 84 — A human epigenome project is now underway in Europe 88 Murrell, this issue , and plans are being hatched for a similar effort in the USA.
These molecular efforts are complemented by advances in epigenomic bioinformatics 80 , 89 , DNA methylation markers hold perhaps even greater promise as detectors of disease, as opposed to classifiers of existing disease Disease detection is useful not only for the early detection of undiagnosed malignancies but also for the monitoring of recurrent disease as a measure of therapeutic efficacy The demands placed on sensitive detection are quite different than those needed for the classification technology.
Sensitive detection requires a high signal-to-noise ratio for the detection of aberrant DNA methylation patterns against a background of normal DNA methylation patterns. In principle, this can consist of the measurement of a single locus, although multiple loci may be needed to achieve sufficient sensitivity and specificity. Sensitive detection technologies tend to rely on sodium bisulfite-based methylation-specific PCR MSP to achieve sufficiently high signal-to-noise ratios Molecular classification, on the other hand, requires that the methylation information is sufficiently complex that subclasses of DNA methylation patterns can be defined.
Hence, the interest in microarray methods and other genome-wide techniques. However, these high-throughput methods are usually based on either methylation-sensitive restriction enzyme digestion or methylation-independent bisulfite PCR, as opposed to MSP, and have modest signal-to-noise ratios. Therefore, classification technologies usually require fairly homogeneous samples, such as primary tumor tissue, and are generally unsuited for sensitive detection purposes. Automated variants of MSP, such as the real-time PCR-based MethyLight technique 92 , have both a sufficient signal-to-noise ratio and the throughput capacity to sensitively analyze a broad set of markers 82 , 93 , The use of DNA methylation markers to detect cancer sensitively is based on the premise that tumor-derived DNA is released into bodily fluids or other remote samples and can be detected by the abnormal DNA methylation patterns specific for malignant cells.
Most studies have used serum or plasma as the source of cell-free DNA However, in recent years, a number of other novel sources of DNA have been used including nipple aspirate fluid 95 , breast fine needle washings 96 , bronchial brush samples 97 , needle biopsies 98 , prostatic fluid or ejaculate 99 , , lymph nodes , , bronchioalveolar lavage , pancreatic juice , sputum , mouth and throat rinsing fluid , exfoliated bladder cells , urine or urine sediments — , peritoneal fluid , , stool 93 and vaginal tampons , As more types of samples are analyzed for a variety of different loci, it is becoming clear that epigenetic changes, including silencing of tumor-suppressor genes, may occur early in malignant progression and can sometimes be detected even in non-malignant or precancerous tissues.
For example, promoter methylation of the p16 INK4a CDKN2A gene is detectable in preinvasive bronchial lesions , in histologically normal human mammary epithelia and in non-adenomatous pituitaries from patients with Cushing's disease Promoter methylation of multiple genes has been reported in non-malignant gastric tissues — , non-neoplastic prostate tissue , , chronic cholecystitis and ulcerative colitis The detection of epigenetic abnormalities in histologically normal or premalignant tissues at risk for progression to malignancy paves the way for the use of DNA methylation markers in risk assessment.
Epigenetic markers should be particularly well suited as risk assessment tools, compared to germline genetic markers, because somatic epigenetic alterations presumably capture lifetime environmental and dietary exposures. Thus, a single class of markers can be used for assessing risk stemming from genotype and environmental exposure, for early detection of malignancy, and for classifying existing disease. In the next few years, the task will be to identify the markers and technologies best suited for each application.
Moreover, if epigenetic changes occur in premalignant tissues, then this opens new avenues for cancer chemoprevention based on the inhibition or reversal of epigenetic alterations before the onset of malignancy The large estimated number of epigenetic alterations found in cancer cells 84 , raises the question whether these reflect stochastic occurrences that accumulate with age, and are selected for during malignant outgrowth, or whether the large number of changes is caused by a defect in one of the components of the epigenetic machinery.
By analogy, both stochastic mutations and mutator phenotypes are involved in producing the genetic alterations found in cancer. Likewise, it is anticipated that both stochastic errors and defects in epigenetic control participate in cancer epigenetics. The identification of these defects will likely emerge in the next few years as we acquire a fuller understanding of the normal mechanisms of maintenance and alteration of epigenetic states. Chromatin protein composition and histone modifications vary throughout the genome, with segregation maintained in part by chromatin insulators and boundaries — West and Fraser, this issue.
Therefore, it is likely that many epigenetic control defects will target a subset of genetic loci, rather than all loci equally.
Likewise, DNMT -deficient cells display distinct subsets of affected loci 24 , — Another nice example of the target specificity of an epigenetic control defect is the increased histone acetylation and transcriptional reactivation of repeat sequences and of the paternally imprinted allele of the Cdkn1c gene in cells deficient for the chromatin remodeling protein Lsh — Recent reports of transcriptional silencing and promoter DNA methylation induced by small interfering RNAs in human cells raise the interesting prospect of an involvement of microRNAs in epigenetic silencing in cancer cells , Mattick, this issue.
One controversial putative epigenetic control defect is the CpG island methylator phenotype CIMP first reported in colorectal cancer , but since described for several other types of cancer as well The source of controversy stems from the fact that only a subset of CpG islands appears to be affected, primarily those that are cancer-specifically methylated.
This is exactly the phenotype that one would predict for a cancer-specific epigenetic control defect, but an extensive study that included a broader analysis of CpG island hypermethylation failed to confirm the existence of CIMP , giving rise to heated debate concerning the existence of CIMP. Other groups have found associations between CIMP and various clinicopathological criteria in colorectal cancer including survival benefit from 5-FU treatment , genetic mutation profiles , location in the right-sided colon and microsatellite instability and association with family history , although this could not be confirmed by another group One such systemic epigenetic control defect appears to be loss of imprinting of the IGF2 locus, which occurs in the colorectal tumors, and in the normal colonic mucosa and white blood cells of individuals with colorectal cancer, or with a family history of colorectal cancer , If epigenetic control defects can occur systemically, then perhaps they can also contribute to cancer risk through transgenerational inheritance.
Promoter methylation of the mismatch repair gene MLH1 is responsible for the majority of colorectal adenocarcinomas with microsatellite instability — This promoter methylation has been found to occur in normal colonic epithelium Recently, strong evidence has accrued that MLH1 methylation can be transmitted through the germline, although true transgenerational inheritance in humans has not yet been formally demonstrated — Such transgenerational epigenetic inheritance has been well documented to occur in mice , Ruden, this issue.
Recently, paramutation, in which one allele can affect the epigenetic state of the other allele, has also been reported to occur in mice It will be interesting to see if the mechanism responsible for this epigenetic cross-talk between alleles contributes to the biallelic CpG island hypermethylation frequently seen in cancer. Mouse models of epigenetic control defects have been particularly useful in demonstrating the important contribution of epigenetics to tumorigenesis. Subsequent work showed that both the size and growth rates of polyps were affected and that a complete suppression of the tumor phenotype could be achieved without drug treatment by using more severe hypomorphic Dnmt1 alleles In other words, intestinal polyp formation in this system is as much dependent on sufficient levels of functional Dnmt1 expression, as it is on the Apc mutation.
These observations are consistent with a model in which polyp formation requires DNA methylation-dependent epigenetic silencing of unidentified tumor-suppressor genes, in addition to loss of heterozygosity of the wild-type Apc allele.
Methyl-binding domain containing proteins MBDs bind to areas of dense DNA methylation and recruit histone deacetylases and transcriptional repressor complexes Fig. As such, MBDs are considered important mediators of epigenetic gene silencing, at the interface between DNA methylation and histone code modification. If the requirement of sufficient Dnmt1 expression for intestinal polyp formation is mediated through epigenetic silencing, then one would anticipate that polyp formation would also depend on other mediators of epigenetic silencing such as MBDs.
However, interestingly, lymphomagenesis is increased in this same model Increased lymphomagenesis in Dnmt1 hypomorphic mice was subsequently confirmed in mice without an Mlh1 mutation Enhanced tumorigenesis in Dnmt1 hypomorphic mice was shown to be related to increased chromosomal instability These findings are consistent with the previously reported increased mutation rates seen in Dnmt1-deficient mouse embryonic stem cells and with the large body of literature describing associations between DNA hypomethylation, particularly of pericentromeric repeats and chromosomal instability in various human experimental and disease models 5 , — However, genomic instability can be attributed to multiple different mechanisms, not all of which may respond to DNA hypomethylation or DNMT1 deficiency in equal ways.
Indeed, others have reported that Dnmt1 deficiency can result in a reduced mutation and deletion rate in embryonic stem cells , consistent with the important contribution of 5-methylcytosine to deamination-mediated mutagenesis in mammalian cells.
The diverse roles that Dnmt1 and DNA methylation play in the maintenance of genomic integrity has been further emphasized by the recent discovery that Dnmt1 is required for efficient DNA mismatch repair and that rates of microsatellite instability increase under Dnmt1-deficient conditions — It is clear from this bird's-eye overview that the field of cancer epigenetics is in flux.
We can expect to see clinical implementation of both epigenetic cancer therapy and epigenetic cancer diagnostics in the next decade. Epigenetic control defects in cancer cells represent an emerging new area of investigation, where significant breakthroughs in the identification of the underlying molecular defects are anticipated in the next few years.
Cancer Epigenetics. Methods and Protocols. Editors: Dumitrescu, Ramona G., Verma, Mukesh (Eds.) Free Preview. Includes epigenetic alterations that can be. Read Cancer Epigenetics: Methods and Protocols (Methods in Molecular Biology ) book reviews & author details and more at curepako.tk Free delivery on.
Epigenetic Silencing. Schematic of some of the molecular events that occur at CpG-rich promoters undergoing epigenetic silencing in cancer cells. The open chromatin structure of a transcriptionally active gene with loosely spaced nucleosomes blue cylinders is shown at the top and the transcriptionally silenced state with more tightly packed nucleosomes is shown at the bottom.
The challenge for the future will be to integrate all these aspects in a complete picture which considers both genotoxicity and epigenetic toxicity in the assessment of the safety of NPs. Top Epigenetics articles: November Audible Download Audio Books. Jackson-Grusby, L. Competing interests The authors declare that they have no competing interests. Kramer, O.
DNA is indicated by a thin black line wrapped around nucleosomes. Proteins are indicated by shaded ovals, histone H3 acetylation is indicated by yellow triangles, hstone H3 methylation is indicated by orange hexagons and CpG dinucleotides are indicated by circles strung along the DNA, with green circles denoting an unmethylated state and red circles indicating a methylated state. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.
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