Cancer Epigenetics

Cancer Epigenetics

C H A P T E R 34 Cancer Epigenetics Kjetil Søreide Stavanger University Hospital, Stavanger, Norway; University of Bergen, Bergen, Norway O U T L I ...

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C H A P T E R

34 Cancer Epigenetics Kjetil Søreide Stavanger University Hospital, Stavanger, Norway; University of Bergen, Bergen, Norway

O U T L I N E Introduction

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Epigenetic Influences Over a Life Time and Cancer Risk

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Mechanisms of Epigenetics in Cancer: Writers, Readers, and Erasers DNA Methylation Histone Modification Noncoding RNA Importance of Epigenetic Changes Together with Genetic Alterations Epigenetics in Intratumoral Heterogeneity and Therapeutic Resistance

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Epigenetic Biomarkers in Cancers Cancer Epigenome in Epidemiology

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Epigenetics as Cancer Therapeutic Targets

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Future Perspectives

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References

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INTRODUCTION Cancer develops through the accumulated changes at the chromosomal level or genetic base pair-level, potentially affecting oncogenes (drivers) or tumor suppressor genes (brakes) that eventually lead to uncontrolled cell division, invasion, and metastasis in various solid organ cancers [1,2]. The mechanisms involved in the hallmarks to cancer development are well described and continues to be explored [3]. Fundamentally, cancer is a genomic disease that alters cellular information flow to modify cellular homeostasis and promote growth, described through mechanisms outlined as the hallmarks of cancer [1,2]. The discovery of a universal genetic code for protein-coding genes produced countless breakthroughs in understanding how such mutations drive cancer, establishing the scientific principles on which the development of targeted therapies for malignancies are based. Thus, maybe one of the most surprising explorations in molecular Handbook of Epigenetics. http://dx.doi.org/10.1016/B978-0-12-805388-1.00034-1 Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic and Epigenetic Classification of Cancer Limitations to Epigenetic Classification and Prognostication

medicine where the discovery that genes where altered in and through noncoding regions, as well as by epigenetic mechanisms. Epigenetics refers to heritable traits that are not attributable to changes in DNA sequence. In more specific terms, it can be used to describe how chromatinassociated proteins and reversible chemical modifications of DNA and histone proteins maintain transcriptional programs by regulating chromatin structures. Epigenetic changes are present in all human cancers and are now known to cooperate with genetic alterations to drive the cancer phenotype through the specific hallmarks of cancer (Fig. 34.1) [3,4]. These epigenetic changes involve mechanisms, such as DNA methylation, histone modifiers and readers, chromatin remodelers, microRNAs (miRNAs), and other components of chromatin [5]. Cancer genetics and epigenetics are inextricably linked in generating the malignant phenotype; epigenetic changes can cause mutations in genes, and, conversely, mutations are frequently

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FIGURE 34.1  Interplay between genomics and epigenomics in cancer. The genome and epigenome influence each other, as the genome provides the primary sequence information and encodes regulators of epigenetic states, whereas the epigenome controls the accessibility and interpretation of the genome. Changes in one can influence the other, forming a partnership in producing genetically or epigenetically encoded phenotypic variation subject to Darwinian selection for growth advantage and thus eventually achieving the hallmarks of cancer [3]. Genetic instability and mutation and epigenomic disruption can be considered enabling characteristics of cancer cells. CIMP, CpG-island methylator phenotype. Source: Reproduced from Shen H, Laird PW. Interplay between the cancer genome and epigenome. Cell 2013;153(1):38–55 [4], with permission from Elsevier.

observed in genes that modify the epigenome [4]. Epigenetic therapies, in which the goal is to reverse these changes, are now entering as standard of care for some types of malignant disease [6,7]. The application of epigenetic therapies in the treatment of solid tumors is also emerging as a potential therapeutic option [8,9]. Notably, epigenetics is not a new field in basic science and epigenetics has been explored since the 1940s [10,11]. However, only more recently has the potential for use as biomarkers and therapeutic options made cancer epigenetics a closer ally for mainstream clinical cancer care only over the past few years [6,12]. Some historic leaps are presented in fig. 34.2, related to several key general epigenetic discoveries and seminal events for epigenetics in colorectal cancer (CRC) [13]. In this chapter we will explore some areas where cancer epigenetics play a role in understanding cancer as a disease, Further, as an example and a well-described public health challenge, CRC will be specifically mentioned as a cancer disease model when suitable, as coverage of all cancer types will be beyond the scope of the chapter. Due to the vast and developing field of research going beyond the scope of this introductory chapter, the

references are chosen mainly for their updated, comprehensive expert review insight. Thus, for the interested reader who wishes to pursue further in-depth knowledge to certain themes, the references provide a direction to sources of detailed knowledge. While specific mechanisms of epigenetics are explored in previous chapters in this book, this chapter overview will largely focus on the areas of immediate or future importance for understanding epigenetics and the role in cancer.

EPIGENETIC INFLUENCES OVER A LIFE TIME AND CANCER RISK Epigenetic studies include the investigation of DNA methylation, histone modifications, chromatin remodeling, and gene regulation by noncoding RNAs (ncRNAs). Epigenetic alterations are critical for early developmental processes, the silencing of the inactive X-chromosome and tissue-specific gene regulation. A comprehensive picture of epigenetic patterns in normal cells is now emerging; these patterns are disturbed in human diseases, such as cancer [14]. Epigenetic marks change

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FIGURE 34.2  A historical perspective illustrating key milestones associated with the discovery of various epigenetic alterations in CRC from 1983 to the present. Individual epigenetic alterations are listed in color-coded boxes, as follows: aberrant DNA methylation (red), ncRNAs, including miRNAs (green), and histone modifications (yellow). CIN, Chromosomal instability; CRC, colorectal cancer; CIMP, CpG-island methylator phenotype; miRNAs, microRNAs; MSI, microsatellite stability. Reproduced from Okugawa Y, Grady WM, Goel A. Epigenetic alterations in colorectal cancer: emerging biomarkers. Gastroenterology 2015;149(5):1204–1225.e12 [13].

during fetal development, through adult life, and with aging [15–18]. Some changes play an important role in the establishment and regulation of gene programs, but others seem to occur without any apparent physiological role. An important future challenge in the field of epigenetics will be to describe how the environment affects epigenetic change and to learn if interaction between various epigenetic influences can determine healthy from those attributed to risk of disease phenotypes during a lifetime. This may give the opportunity for potential risk-reducing interventions, life-style changes, or even epigenetic preventive therapy. Chemical and physical environmental stressors, diet, life habits, and pharmacological treatments can affect the epigenome [15,19]. The potential use of natural epigenetic modifiers in the chemoprevention of cancer to link together public health, environment, and lifestyle is increasingly explored and has gained interest from a public health perspective and for prevention of disease [20]. The way in which energy is used in cells is determined under the influence of environmental factors, such as nutritional availability [21]. Metabolic adaptation is mainly achieved through the modulation of metabolic gene expression [22,23], and may also involve epigenetic mechanisms that enable long-term regulation. Recent studies have identified that nutrients and their metabolites exert an important influence on the epigenome, as

they serve as substrates and/or coenzymes for epigenetic-modifying enzymes. Some epigenetic factors have been shown to regulate metabolic genes leading to a shift in energy flow. These findings suggest the concept of metabolism-epigenome cross-talk that may contribute to the formation of a long-term metabolic phenotype [21]. This is particularly relevant to the pathogenesis of obesity and associated metabolic disorders, in which pre- and postnatal nutritional conditions affect disease risks in adulthood. Further, the aging human colorectal mucosa develops aberrant patterns of DNA methylation that may contribute to its increasing vulnerability to cancer [24]. Evidence suggest that age-dependent loss of global methylation, together with hypermethylation of CpG islands associated with cancer-related genes, may be influenced by nutritional and metabolic factors [25]. Several compounds of nutrition, such as folates and vitamins, are essential for the maintenance of normal DNA methylation. Folate metabolism is known to modify epigenetic mechanisms under experimental conditions, and more recent findings has explored the important roles of vitamin C and D in maintenance of the epigenome [26,27]. Human intervention trials and cross-sectional studies suggest a role for folates and other nutritional and metabolic factors as determinants of colorectal mucosal DNA methylation [24]. Moreover, most cancer cells exploit metabolic pathways for their

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hyperproliferative activity [28] while metabolic dysregulation leads to aberrant epigenetic regulation in some cancers [29].

MECHANISMS OF EPIGENETICS IN CANCER: WRITERS, READERS, AND ERASERS Genetic mechanisms of mutation, copy number alteration, insertions, deletions, and recombination are particularly well suited as vehicles of persistent phenotypic changes in cancer. Several past cancer studies investigated this and developed sequential progression models, such as that presented for CRC [30]. For this reason, cancer has long been viewed as a genetic disease, although different methylation profiles were noted to occur in different genes in the same tumor model [31].

Knowingly, genetic events occur at low frequency and are thus not a particularly efficient means for malignant transformation [32]. However, some cancer cells overcome this bottleneck by acquiring DNA repair defects, thus boosting the mutation rate such as seen in mismatch repair deficient tumors that lead to high occurrence of microsatellite instability (MSI) throughout the genome [33,34]. Mechanisms of epigenetic control offer an alternative path to acquiring stable oncogenic traits [4,35]. Epigenetic states are flexible yet persist through multiple cell divisions and exert clear-cut effects on the cellular phenotype. Although cancer cells have long been known to undergo epigenetic changes, genome-scale genomic and epigenomic analyses have only recently revealed the widespread occurrence of mutations in (epi)genetic regulators and the breadth of alterations to the epigenome in cancer cells. There are several classes of epigenetic regulators (Fig. 34.3), those that write the marks (dubbed

FIGURE 34.3  Epigenetic writers, erasers, and readers. The basic functional unit of chromatin is the nucleosome, which is composed of DNA wrapped around histones (H2A, H2B, H3, and H4). Core histone tails are projected from nucleosomes and are subject to PTMs. These include Me, Ac, Ph, and Ub. The main epigenetic regulators can be categorized as writers, erasers, and readers of PTMs. Epigenetic writers are responsible for the addition of chemical modifications. Epigenetic erasers catalyze the removal of the covalent modifications. Epigenetic readers are proteins with specific domains that recognize and bind to particular modifications. Ac, Acetylation; MBT, malignant brain tumor; Me, methylation; Ph, phosphorylation; PTMs, posttranslational modifications; Ub, ubiquitination. Reproduced from Simó-Riudalbas L, Esteller M. Targeting the histone orthography of cancer: drugs for writers, erasers and readers. Br J Pharmacol 2015;172(11):2716–32 [8].

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Mechanisms of Epigenetics in Cancer: Writers, Readers, and Erasers

writers), DNA methyltransferases, histone methyltransferases (HMTs), and histone acetyltransferases (HATs); those that read the marks (dubbed readers), the bromodomain, chromodomain, and tudor proteins; those that erase the marks (dubbed erasers), histone deacetylases (HDAC) and histone demethylases (HDM); and those that remodel the chromatin, such as components of the SWI/SNF complex [8,36]. The frequent, recurrent mutation of specific epigenetic modifiers in a variety of cancers demonstrates that altered epigenetic regulation plays an important role in driving tumorigenesis. While epigenetics represents a wide range of changes that regulate gene expression, yet does not cause changes in the primary base-pair sequence of the DNA in itself, these can be broadly defined as DNA methylation, histone modifications, genome imprinting, and noncoding RNA (ncRNA). Previous chapters have detailed mechanistic insights to each of these mechanisms, so only a brief presentation related to cancerogenesis will be discussed.

DNA Methylation The best-known and most explored epigenetic marker/mechanism is DNA methylation [10]. DNA methylation occurs in the context of chemical modifications by the addition of a methyl group to DNA at the 5-carbon of the cytosine pyrimidine ring that precedes a guanine. DNA methylation has critical roles in the control of gene activity and the architecture of the nucleus of the cell. Hypo- and hypermethylation will consequently alter the access to and expression of certain genes or DNA parts. Further, histones are not merely DNA-packaging proteins, but molecular structures that participate in the regulation of gene expression and, thus, their chemical modification may also alter these functions. Histones store epigenetic information through such posttranslational modifications as lysine acetylation, arginine and lysine methylation, and serine phosphorylation. These modifications affect gene transcription and DNA repair [13], as depicted in Fig. 34.4A. In humans, DNA methylation occurs in cytosines (C) that precede guanines (G); these are called dinucleotide “CpGs.” Regions in the DNA that contain many adjacent C and G nucleotides are called “CpG-islands.” The “p” in CpG refers to the phosphodiester bond between the cytosine and the guanine. These islands occur in approximately 40% of the promoters of human genes. CpG sites are not randomly distributed in the genome; instead, there are CpG-rich regions (CpG islands), which span the 5′ end of the regulatory region of many genes. These CpG-islands are usually not methylated in normal cells. While CpG islands are usually unmethylated in normal cells, and the genes downstream of these unmethylated promoters are transcribed in the presence of transcriptional activators, genomic platforms have

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confirmed that almost 10% of normally unmethylated promoter CpG islands, many of them belonging to tumor suppressor genes, become abnormally methylated and thus silenced in cancer [2,11]. In CRC [34,37], a CpG-island methylator phenotype (CIMP) has been recognized as a separate molecular group (besides microsatellite instable and chromosomal instable tumors) that has distinct clinical and pathological characteristics. For one, prognosis is demonstrated to be consistently poorer with the CIMP group [38].

Histone Modification Heterochromatin is a closed chromatin conformation that is, often associated with DNA methylation and inactive gene transcription (Fig. 34.4B). In contrast, the euchromatin state is in an open conformation and associates with active gene transcription, presumably secondary to increased transcription factor binding. DNA methyltransferases and MBDs work with histonemodifying enzymes for the purpose of regulating all DNA-templated processes including transcription, repair, replication, and recombination [10,11,39]. Histone N-terminal tails can undergo many chemical modifications, including acetylation, methylation, phosphorylation, and ubiquitination (see Fig. 34.4B). Depending on the particular combination of modifications in a specific genomic region, chromatin remains more or less packed, blocking, or permitting the nuclear processes. Importantly, this histone code is not static but rather is changing in a context-dependent manner to facilitate or repress, for instance, gene transcription. New histone modifications are still being discovered and are found in new combinations. The influence of these marks on the deposition, interpretation, and erasure of other histone modifications is known as histone cross-talk (Fig. 34.5) and is of great importance for the transcriptional readout of a gene [11,36]. The enzymes responsible for such modifications throughout histone tails include HATs, HDACs and sirtuins, HMTs, HDMs, histone kinases and phosphatases, histone ubiquitin ligases, and deubiquitinases (as reviewed in detail in Ref. [11]). Consistently, with the fact that disruption of normal patterns of covalent histone modifications is a defined hallmark of human cancer many such enzymes that add or remove these chemical groups, and also those that recognize them (the readers), are mutated or misregulated in cancer.

Noncoding RNA Although the most thoroughly studied sequences in the human genome are those of protein-coding genes, these translated regions account for only 2% of the entire DNA. The remaining transcribed but nonprotein-

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FIGURE 34.4  An illustration of various epigenetic alterations in CRC. (A) The concept of aberrant DNA hypermethylation in the context of a “cancer cell” is shown. Double helix DNA represents a tumor suppressor gene with CpG islands and CpG shores in its promoter region. Hypermethylation of CpG sites (black “lollipops”) leading to gene silencing and closed chromatin in the cancer cells is shown. In contrast, CpG dinucleotides within introns, as well as in intergenic regions are frequently hypomethylated, which may lead to the increased expression of oncogenes and oncomiRNAs, and resulting open chromatin conformation. (B) Histone modifications in a cancer cell. (Left) Heterochromatin, which is a closed chromatin conformation that is, often associated with DNA methylation and inactive gene transcription. In contrast, the euchromatin state is in an open conformation and associates with active gene transcription, presumably secondary to increased transcription factor binding. HDAC, Histone deacetylases (C) Schematic demonstrates miRNA biogenesis in cancer cells and how miRNAs inhibit and/or cause degradation of their mRNA targets. (D) Illustration of various activities of long, noncoding RNAs (lncRNAs) in cancer cells, including their abilites (i) to regulate chromatin conformation; (ii) to induce transcription by binding to appropriate transcription factors; (iii) to function as decoys and inhibit gene transcription; (iv) to act as miRNA sponges; (v) to cause mRNA decay; and (vi) to induce mRNA translation. Reproduced from Okugawa Y, Grady WM, Goel A. Epigenetic alterations in colorectal cancer: emerging biomarkers. Gastroenterology 2015;149(5):1204–1225.e12 [13].



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FIGURE 34.5  Hierarchical organization of epigenetic regulation. Dynamic and reversible epigenetic processes generate diverse regulatory environment. DNA methylation (1) (mC, methylcytosine, represented by closed circle) may result in eviction of DNA-binding proteins, or recruitment of methyl-binding factors. mC oxidation (shown as hydroxymethylcytosine, hmC) generates additional diversity. Core and linker histone exchange, including variant histone incorporation (2), regulates local DNA accessibility and, together with histone modifications (3), introduces local variations to chromatin structure. These are interpreted by the “reader” machinery (3) and drive higher-order chromatin organization and nuclear topology (4). Red arrows indicate cross-talk between regulatory layers. Reproduced in Soshnev AA, Josefowicz SZ, Allis CD. Greater than the sum of parts: complexity of the dynamic epigenome. Mol Cell 2016 ;62(5):681–94 [36], with permission from Elsevier.

coding part of the genome is made up of ncRNAs, which are increasingly recognized as being fundamental to embryogenesis and development and are also disrupted in diseases, such as cancer [11,40]. The ncRNAs include miRNAs, PIWI-interacting RNAs (piRNas), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs). Illustration of their role is given in Fig. 34.3C (miRNA) and Fig. 34.3D (lncRNA). Extensive reviews are presented elsewhere on these topics [40–42].

Importance of Epigenetic Changes Together with Genetic Alterations Notably, tumors evolve in three broad phases, as reviewed by Vogelstein and Kinzler [43]. In the breakthrough phase, a cell acquires a driver-gene mutation and begins to proliferate abnormally [43]. It takes many cell divisions, and many years, for the cells resulting from this proliferation to be observable clinically, if they ever are. For example, nevi on the skin and small adenomas in the intestine are detected only because they can be easily observed on visual inspection or colonoscopy. Similarly sized lesions in internal organs (e.g., kidneys or the pancreas) would generally be undetectable clinically. The expansion phase is driven by a second drivergene mutation enabling the cell to thrive in its local environment despite low concentrations of growth factors, nutrients, oxygen, and appropriate cell-to-cell contacts [43]. Known cancer mutation rates suggest that this second mutation is unlikely to occur absent of a large increase in cell number during the breakthrough phase

[32]. The likelihood of progression thus depends on both the mutation rate and the number of cells at risk for acquiring another mutation. The first two mutations lead to the abnormal proliferation and disordered cellular architecture defining benign tumors. Subsequent mutations enable cells to invade normal tissues and grow in otherwise hostile environments; such cells are by definition malignant. The mutation initiating the breakthrough phase is often very specific, a limited number of growth-regulating pathways seem able to initiate neoplasia in a given cell type. As tumors progress, this specificity seems to be progressively lost, so a greater number of driver genes can transform a cell from the expansion phase to the invasive phase (see Ref. [43] for overview). The fact that so few genetic mutations are required in neoplastic transformation leaves several unanswered questions. This “dark matter,” as alluded to [32], is most likely controlled and influenced by epigenetic mechanisms. Genome-wide sequencing has shown that every tumor harbors thousands of genetic (and epigenetic) alterations that are not present in the patient’s germline. Of notice, only a very small fraction of these alterations are in “driver genes” — genes that when mutated endow the tumor cell with a growth advantage over surrounding cells [32]. The remaining alterations are “passengers,” found in tumor cells only because they occurred coincidentally during the long march toward tumorigenesis. Only about 200 of the 20,000 genes in the human genome have been shown to act as driver genes for common cancers [32,43]. Moreover, these genes appear to function through a limited number of pathways that

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regulate cells’ growth and fate. Thus, the implications of genes in tumor initiation may be reduced to a few general principles in cancer development from preneoplasia to invasive cancer. How epigenetics involve genetics in carcinogenesis (and vice versa) is not fully understood, other than the two being mutually involved [4,44]. Notably, techniques in genetics research has expanded tremendously over the past decades, enabling a more rapid and widespread investigation into genetic disruptions. At the same time, epigenetics research has largely focused on DNA methylation, as this feature is the most stable mark that survives various sample processing, including DNA extraction and even formalin fixation and paraffin embedding [45]. However, mechanisms and relations related to epigenetic reprogramming in cancer cells, roles in cell plasticity, and role for intratumor heterogeneity are being explored, of which some relations are briefly mentioned here. Epigenetic reprogramming of neoplastic cells have been proposed [46], in that by activating specific transcription factor drivers and modulating collaborating chromatin regulators, cancer cells may dynamically regulate their epigenetic circuits to rewire differentiated cancer cells into stem-like cells (Fig. 34.6), thus refueling cancer growth [46]. While the idea of stem-cells or “stemness” remains a controversial topic in and of itself, the idea and theory of this cellular plasticity model is highly entertained as a mechanism for both understanding cancer development and as a mode for potential intervention [47–49]. An epigenetic plasticity of tumor cells have been proposed to be fundamental for tumor cells to be able to metastasize [50]. Cancer stem cells (CSCs) have been identified in various tumors and are defined by their

potential to initiate tumors upon transplantation, selfrenewal, and reconstitution of tumor heterogeneity [51]. Modifications of the epigenome can favor tumor initiation by affecting genome integrity, DNA repair, and tumor cell plasticity. Importantly, an indepth understanding of the epigenomic alterations underlying neoplastic transformation may open new avenues for chromatin-targeted cancer treatment, as these epigenetic changes could be inherently more amenable to inhibition and reversal than hard-wired genomic alterations.

Epigenetics in Intratumoral Heterogeneity and Therapeutic Resistance By the time of diagnosis, any patient’s tumor may consist of tens of thousands or even millions of cells that have differentiated and created several subclones within the tumor. This heterogeneous mixture of functionally distinct cancer cells can be caused by varying levels of receptor activity, differentiation, and distinct metabolic and epigenetic states. Several classes of epigenetic regulators (Fig. 34.3) of writers, readers, and erasers have been implicated in mechanisms leading to intratumoral heterogeneity and chemotherapy resistance [49,52,53]. The clinical implications are several fold, and mechanism are explored and knowledge is expanding. First, knowledge and ability to biopsy tumor changes in subclones at the time of diagnosis and, as well during follow up may help tailor the appropriate therapy to the epigenetic and genetic makeup of the tumor. Further, epigenetic marks may be used as biomarker of treatment success; either for measure of response or as indicator of recurrence. Finally, specific epigenetic drugs may be used together

FIGURE 34.6  Cellular hierarchies in normal tissues and malignancies. Normal tissues (left) and a growing list of malignancies (right) have established epigenetic hierarchy, with rare populations of stem cells giving rise to more differentiated cellular progeny through intermediate steps (color shades). Reprogramming experiments have shown that differentiation is reversible (left and right arrows). Cellular transformation (red arrow) is a stepwise process involving accumulation of genetic and epigenetic hits. Once initiated, additional and potentially divergent alterations may occur, establishing a tumor with genetic heterogeneity (illustrated by yellow and green stars) and, within each genetic subclone, an epigenetic hierarchy (color shades). Altered activity of key regulators, including CRs and TFs, can play dual roles in cancer, contributing to transformation and epigenetic state transitions (oncogenic reprogramming). We speculate that the same network of regulators may then act within the established tumor to rewire differentiated cancer cells into stemlike cells, thus establishing a dynamic equilibrium between differentiation and reprogramming. CR, Cromatin regulator; TF, transcription factor.

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Genetic and Epigenetic Classification of Cancer

with conventional drugs to achieve a more tailored and specific response to the tumor at hand. Several such principles are reviewed extensively elsewhere [6,49,52,53].

GENETIC AND EPIGENETIC CLASSIFICATION OF CANCER Tumors have historically been classified based on their (likely) tissue of origin and differentiation and grade. This still represents the mainstay for diagnosis and prognosis, but increasingly the molecular features help classify tumors into distinct therapeutic and prognostic classes. In CRC, the tumor-node-metastasis

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(TNM) system is still used for prognosis and therapeutic decisions, but it is recognized to have several limitations with risk of over- and undertreatment [54]. Also, it does not capture the specific underlying genetic pathways involved, although these are to some degree incorporated into routine testing for a few select mutations (e.g., RAS mutation status for EGFR treatment) [55]. Commonly observed alterations across sporadic CRCs have now allowed classification of CRC into at least 3 possible distinct groups [34] and emerging markers of good, bad and ugly disease behavior is proposed for advanced disease (e.g., liver metastases) [56]. For the assessment of primary CRCs (Fig. 34.7) there are at least 3 suggested classifications that come into consideration.

FIGURE 34.7  Timeline for sporadic CRC pathogenesis and its characterized molecular pathways. (A) The timelines are based on the mean age of CRC for each type. Tumorigenesis can be broken into tumor initiation (development of an adenoma), tumor progression that culminates in a malignancy (carcinoma) that can spread as metastasis. MSI-H tumors are known to have a shortened progression stage. (B) Each pathway has its feature of moving from normal to cancer and potentially metastasis, with varying histology. Wnt signaling is the gatekeeper for all 3 pathways. The CIMP pathway contributes to both the MSI-H (through hypermethylation of hMLH1) and CIN pathways, and specifically characterizes a serrated pathway. EMAST can modulate all 3 pathways. Reproduced with permission from Carethers JM, Jung BH. Genetics and Genetic biomarkers in sporadic colorectal cancer. Gastroenterology. 2015;149(5):1177–1190.e3 [34].

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One is a hypermutated group that includes defective DNA mismatch repair with MSI and POLE mutations (about ∼15% of CRC patients), containing multiple frameshifted genes and BRAFV600E. The second is a nonhypermutated group with multiple somatic copy number alterations and aneuploidy, previously known as chromosomal instability (CIN) type of tumors (in ∼85%), containing oncogenic activation of KRAS and PIK3CA and mutation and loss of heterozygosity of tumor suppressor genes, such as APC and TP53. A third group is named CpG island methylator phenotype (CIMP) type CRCs (in ∼20%) that overlap greatly with MSI CRCs and some nonhypermutated CRCs. CIMP tumors have methylated CpG islands and epigenetic alterations are essential in these cancers. Lastly a fourth group (or, maybe more appropriate; a modifier group or trait occurring across all other groups) is named after Elevated Microsatellite Alterations at Selected Tetranucleotide (EMAST) repeats (found in up to ∼60% of CRC, but also in a range of other cancer types [33]) that associates with metastatic behavior in both hypermutated and nonhypermutated groups. Components from these classifications are now used as diagnostic, prognostic, and treatment biomarkers [34], yet universal agreement on such a new classification has not been reached with alternative proposals published [57,58]. It should also be noted that evidence from the EMAST classification is not yet clear [33], although studies are emerging [59–61]. Additional common biomarkers may come from genome-wide association studies and miRNAs, among other sources, as well as from the unique alteration profile of an individual CRC to apply a precision medicine approach to modern cancer care. The delineation of molecular markers in CRC metastasis (Fig. 34.8) is also becoming of age and may play an even greater role with advancement in epigenetic therapies in the future [62]. As an example, clinical behavior of CRC is determined by several factors, including demographic data (age, gender, race) and tumor presentation (location, stage) and timing of presentation of metastasis (synchronous or metachronous), as depicted in Fig. 34.8A. Embedded in the cancer cells are the molecular pathways, which follows distinct forms of genomic instability yet with partly overlapping areas. Hypermutated cancers belong to the microsatellite instable (MSI) cancers and in part the CIMP cancers. Nonhypermutated follow in large parts the chromosomal instability (CIN)–driven pathways, often involving KRAS mutations from an early stage. The propensity to develop metastasis may possibly be modified through the elevated microsatellite alterations at selected tetranucleotide repeat (EMAST) and associated mechanisms, such as regulation of miRNAs or activity and numbers of CD8+ immune cells. Finally, the microenvironment contains

numerous factors that may facilitate or propagate metastasis to invade, spread and settle in a new organ sites, particularly the liver and the lungs. Determined by the clinical presentation (Fig. 34.8B), the genetic traits and molecular mechanisms, the prognosis in colorectal liver metastasis is related to resectabilty for long-term survival. “Good” cases amenable for surgery have fewer bad genetic traits, such as less likelihood for BRAF mutations or KRAS mutations, and are more likely to have EMAST and low-frequency MSI-L, as demonstraed by Koi et al. [63]. Patients with concomitant liver and lung metastases have an “ugly” tumor biology and are more likely to have higher frequencies of both KRAS and BRAF mutations and respond poorly to any line of treatment. The “bad” cases are in between, and the shift from “nonresectable” to “resectable” experiences a positive drift with time and where changing practice in surgical strategy, novel techniques and use of conversion chemotherapy regimens improve outcomes. Novel biomarkers may aid in understanding aggressiveness of liver metastasis, assist in clinical decision making and help to find new and more efficient therapies.

Limitations to Epigenetic Classification and Prognostication Generalisability and reproducibility in using epigenetic markers for classification and diagnosis has been hampered by the lack of standardized and unified protocols and analytical designs. For example, we found in a study that the call of CIMP classification would deviate substantially between cases depending on what definitions, genes and panels where used for defining CIMP status [64]. A systematic review examined all published studies on CRC prognosis according to the different definitions of CIMP and identified 36 studies [65]. Among these, 30 (83%) studies reported the association of CIMP and CRC prognosis and 11 (31%) studies reported the association of CIMP with survival after chemotherapy. Overall, 16 different definitions of CIMP were identified [65]. The majority of studies reported a poorer prognosis for patients with CIMP-positive CRC than with CIMP-negative CRC. Inconsistent results or varying effect strengths could not be explained by different CIMP definitions used. No consistent variation in response to specific therapies according to CIMP status was found. As the authors conclude, comparative analyses of different CIMP panels in the same large study populations are needed to further clarify the role of CIMP definitions and to find out how methylation information can best be used to predict CRC prognosis and response to specific CRC therapies. From both studies [64,65] goes the notion that better standardization and agreement between studies are needed.

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FIGURE 34.8  Clinical and molecular influence on aggressiveness of colorectal liver metastasis. (A) Clinical behavior of CRC is determined by several factors, including demographic data (age, gender, race) and tumor presentation (location, stage) and timing of presentation of metastasis (synchronous or metachronous). Embedded in the cancer cells are the molecular pathways, which follows distinct forms of genomic instability yet with partly overlapping areas. Hypermutated cancers belong to the microsatellite instable (MSI) cancers and in part the CIMP cancers. Nonhypermutated follow in large parts the CIN–driven pathways, often involving KRAS mutations from an early stage. The propensity to develop metastasis may possibly be modified through the elevated microsatellite alterations at selected tetranucleotide repeat (EMAST) and associated epigenetic mechanisms, such as regulation of miRNAs or activity and numbers and activity of CD8+ immune cells. Finally, the microenvironment contains numerous factors that may facilitate or propagate metastasis to invade, spread and settle in a new organ sites, particularly the liver and the lungs. (B) Determined by the clinical presentation, the genetic traits and molecular mechanisms, the prognosis in colorectal liver metastasis is related to resectabilty for long-term survival. “Good”’ cases amenable for surgery have fewer bad genetic traits. Patients with concomitant liver and lung metastases have an “ugly” tumor biology and are more likely to have higher frequencies of both KRAS and BRAF mutations and respond poorly to any line of treatment. The “bad” cases are in between, and the shift from “nonresectable” to “resectable” experiences a positive drift with time and where changing practice in surgical strategy, novel techniques and use of conversion chemotherapy regimens improve outcomes. Reproduced with permisission from Søreide K, Watson MM, Hagland HR. Deciphering the Molecular Code to Colorectal Liver Metastasis Biology Through Microsatellite Alterations and Allelic Loss: The good, the bad, and the ugly. Gastroenterology. 2016;150(4):811–4 [56].

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EPIGENETIC BIOMARKERS IN CANCER The ability to identify high- and low-risk patients in cancer beyond the regularly used TNM system, is still somewhat poor [54]. Molecular biology has, over the years, given insight into basic principles of cancer initiation and development and increasingly these are being exploited as biomarkers, and several epigenetic markers or tools have been proposed in various tumors. This include aberrations increasing risk of tumor development, (epi-)genetic changes associated with the stepwise progression of the disease, and errors predicting response to a specific treatment. As several epigenetic changes occur before histopathological changes are present, they can serve as biomarkers for cancer diagnosis and risk assessment [66]. Many cancers may remain asymptomatic until relatively late stages; in managing the disease, efforts should be focused on early detection, accurate prediction of disease progression, and frequent monitoring [13]. Based on epigenomic information, several biomarkers have been identified that may serve as diagnostic tools; such biomarkers also may be useful in identifying individuals who will respond to therapy and, potentially, live longer [2,13,34,56,66–68]. As techniques develop and investigations expand, the putative list of potential epigenetic biomarkers grow longer. However, methylation status is a (if not the most) frequently investigated marker [69], as indicated by the CpG-island methylation status explored for CRC. Further, ncRNAs may serve as biomarkers, exemplified as miRNA profiles. However, as the average CRC methylome has hundreds to thousands of abnormally methylated genes and dozens of altered miRNAs [13,70], it is hard to define any given marker as valid. Further, biomarker validity and robustness depends on stage of disease (e.g., preneoplasia, early stage or, metastatatic situation), will likely be dependent on a set of markers (e.g., related to other genetic mutations or, for example, as set of miRNA alterations) and, is dependent on the outcome as endpoint (i.e., whether it is suited for diagnosis, prognosis, or therapy prediction) [71]. Thus, much is still to be learnt for the best use and exact role of epigenetic markers in cancer disease outcome prediction. Much like the genome-wide association studies (GWAS) studies, EWAS are likely to require large international consortium-based approaches to reach the numbers of subjects, and statistical and scientific rigor, required for robust findings [72,73].

Cancer Epigenome in Epidemiology Epigenetic epidemiology includes the study of variation in epigenetic traits and the risk of disease in populations. Its application to the field of cancer has provided insight into how lifestyle and environmental factors influence the epigenome and how epigenetic events

may be involved in carcinogenesis [74]. Furthermore, it has the potential to bring benefit to patients through the identification of diagnostic markers that enable the early detection of disease and prognostic markers that can inform upon appropriate treatment strategies. However, there are a number of challenges associated with the conduct of such studies, and with the identification of biomarkers that can be applied to the clinical setting [74]. Completion of the human genome a decade ago laid the foundation for using genetic information in assessing risk to identify individuals and populations that are likely to develop cancer, and designing treatments tailored to a person’s genetic profile (coining the term precision medicine). GWAS completed during the past several years have identified risk-associated single nucleotide polymorphisms (SNPs) that can be used as screening tools in epidemiologic studies of a variety of tumor types. Just as GWAS grew from the field of genetic epidemiology, so too do epigenome-wide association studies (EWAS) derive from the burgeoning field of epigenetic epidemiology, with both aiming to understand the molecular basis for disease risk [73]. EWAS to identify regulatory mechanisms and to predict their causal link to common diseases currently make use of a crude proxy by examining percentage methylation differences at ∼2% of CpG sites in the genome, sites selected in part for their accessibility using available detection methods [69,75]. Now, novel pipelines in which candidate functional SNPs are first evaluated by fine mapping, epigenomic profiling, and epigenome editing, and then interrogated for causal function by using genome editing to create isogenic cell lines followed by phenotypic characterization are reported [76], which may generate new insight into causality and relations between genetics and epigenetics and relation to disease risk. While genetic risk of disease is currently unmodifiable, there is, at least in theory, a potential for that epigenetic risk may be reversible and or modifiable through modifiers. There have been numerous diseases, exposures and lifestyle factors investigated with EWAS, with several significant associations now identified (reviewed in [72,73]). Currently, it has been difficult for researchers to understand disease risk from GWAS results [77], as most GWAS-identified SNPs are located in noncoding regions of the genome. Thus, the GWAS field has been left with the conundrum as to how a single-nucleotide change in a noncoding region could confer increased risk for a specific disease. One possible answer to the causality puzzle is that the variant SNPs cause changes in gene expression levels rather than causing changes in protein function [77]. The current hypothesis is that one or more of these risk-associated noncoding SNPs cause changes in gene expression of a critical gene [77]. However, functional follow-up experiments are both expensive and time-consuming and one cannot test each possible

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candidate SNP for causality. Many of the SNPs found in noncoding regions are in relation to promoters and enhancers. While investigators can bioinformatically identify promoter SNPs, it is more diffcult to identify SNPs within enhancers [77]. Unlike promoters, the enhancers do not occur at a defined distance from a transcription start site (TSS). However, they can be identified by specific epigenomic profiles [77]. Several methodological and biological implications are drawn from past findings with the need for improved technology, analyses, and interpretations (as discussed in detail in [77]). However, it is hoped that information gained from GWAS and EWAS will have potential for applications in cancer control and treatment in the future [72,73]. However, much like the GWAS studies, EWAS are likely to require large international consortium-based approaches to reach the numbers of subjects, and statistical and scientific rigor, required for robust findings.

EPIGENETICS AS CANCER THERAPEUTIC TARGETS Genetic mutations and gross structural defects in the DNA sequence permanently alter genetic loci in ways that significantly disrupt gene function. In contrast, genes modified by aberrant epigenetic modifications remain structurally intact and are thus subject to partial or complete reversal of modifications that restore the original (i.e., nondiseased) state. Such reversibility makes epigenetic modifications ideal targets for therapeutic intervention [8]. For a comprehensive presentation of targets of epigenetic writers, readers, and erasers the reader is referred to a recent update on this comprehensive topic [8]. Notably, epigenetic therapy is currently entertained in few malignant diseases, most notably in a preleuke mic disorder called myelodysplastic syndrome (MDS) and in acute myelogenic leukemia (AML) using DNA hypomethylating agents (HMA) [78,79]. The role of epigenetics on solid organ cancers is still early days and awaits further trials [62,80,81]. Several other epigenetic regulating agents are undergoing trials, including HDAC inhibitors (HDACi) [82], currently under development for use as anticancer agents following the FDA approval of two HDACis, namely vorinostat and romidepsin [83]. The areas that are being explored are epigenetic sensitization to radiotherapy, epigenetic sensitization to cytotoxic chemotherapy, and epigenetic immune modulation and priming for immune therapy [62,84–86]. Emerging therapies targeting specific epigenetic modifications or epigenetic modifying enzymes either alone or in combination with other treatment regimens are now extensively being studied for hematologic malignancies [78,79] and solid cancer types [62,87] alike. Several new mechanisms and possible early phase trials are discussed

in more detail elsewhere [8,62,80,81,88–92]. The limitations posed by cancer treatments involve (among others) the unintended epigenetic modifications that may result in exacerbation of tumor progression–a side effect that clearly would contradict the use of epigenetic therapy for curative purposes. The specificity restrictions (i.e., tumor specific effect not involving the normal epigenome) posed by epigenetic therapies and ways to address such limitations is presented in detail elsewhere [93]. Further, with the next generation of targets and drugs, there is a hope that novel epigenetic therapies may improve drug targeting and drug delivery, optimize dosing schedules, and improve the efficacy of preexisting treatment modalities, such as chemotherapy, radiation, and immunotherapy [90].

FUTURE PERSPECTIVES For cancer as a genetic disease, considerable developments have occurred over the past decades with insights into a diverse collection of biological phenomena that cannot be explained by genetics alone. It is becoming clear that a wide range of epigenetic pathways, affecting nucleic acids and histone proteins, are dynamic and reversible, offering considerable promise for a better understanding of normal biology, pathophysiological processes, and with the potential for treatment of human disease, such as cancer [35,36,94,95]. These insights have had a paradigm-shift impact on our understanding of normal and perturbed development. Indeed, the dynamic epigenome adds an additional layer of complexity to the function of our genome, leading to “a sum greater than its parts” [36]. As such, advances in genome editing, visualization technology, and genomewide analyses have revealed unprecedented complexity of chromatin pathways, offering explanations to longstanding questions and presenting new challenges [36]. The combined investigation of GWAS and EWAS studies may yield better determination of risk factors for improved cancer control [72]; the advances in epigenetic therapies may render new therapy options for radiationand chemotherapy-refractory neoplasia with no other current valid treatment options [96]; and, for other cancer types, it may yield a wider range of options tailored to the underlying (epi-)genetic mechanisms of the cancer for personalized cancer therapy [97,98].

Abbreviations CIMP CIN CR CRC CSC EWAS

CpG island methylator phenotype Chromosomal instability Cromatin regulator Colorectal cancer Cancer stem cells Epigenome-wide association studies

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GWAS Genome-wide association studies HAT Histone acetyltransferases HDAC Histone deacetylases HDM Histone demethylases HMT Histone methyltransferases lncRNA Long noncoding RNA miRNA MicroRNAs MSI Microsatellite instability ncRNA Noncoding RNA piRNa PIWI-interacting RNA PTM Posttranslational modification siRNA Small interfering RNA snoRNA Small nucleolar RNA SNP Single nucleotide polymorphisms TF Transcription factor TNM Tumor-node-metastasis system

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