**1. Introduction**

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths globally and is expected to be responsible for an estimated 1.1 million deaths by 2030 [1]. With this growing global burden, prevention and treatment of CRC remains a significant public health challenge. CRC is thought to originate from sequential accumulation of genetic and epigenetic aberrations [2]. Of the identified genetic mutations, approximately 15 have been characterized as "driver mutations" and are thought to be functionally important during CRC initiation and progression [3, 4]. These include genes affecting critical cellular pathways such as those governing proliferation, apoptosis, migration, adhesion, and DNA damage and repair [3]. Importantly, it is now well established that epigenetic alterations can also serve as major driver events in the pathogenesis of CRC [5–7]. However, unlike genetic mutations, epigenetic modifications consist of heritable changes in gene expression without DNA sequence changes and are intrinsically reversible by nature. These epigenetic events include alterations in DNA methylation, histone modifications, and non-coding RNAs. Moreover, the reversibility of these modifications makes them attractive molecular targets for anticancer therapeutic interventions [3, 8].

CRC is a highly heterogeneous disease and can be classified into molecularly and pathologically distinct pathways and subtypes [9]. Moreover, these classifications have significantly influenced patient stratification, prognosis, and therapeutic response [9, 10]. In this chapter, we focus on three epigeneticrelated primary molecular pathways, namely the microsatellite instability (MSI) phenotype, the chromosomal instability (CIN) phenotype, and the CpG island methylator phenotype (CIMP). Importantly, each pathway reflects the underlying mechanisms of carcinogenesis as marked by certain aberrations such as a defective DNA mismatch repair (MMR) system, which is associated with MSI CRCs [11], or by widespread promoter DNA methylation within CpG islands as is the case with CIMP tumors [11, 12]. On the other hand, the CIN pathway, which manifests in majority of CRC cases (~85%), arises through widespread chromosomal imbalances [9, 13, 14]. We also make mention of the relationship between these defined pathways and the four consensus molecular subtyping classifications, with emphasis on the frequent overlap observed between two or more of the aforementioned pathways.

In the past few decades, several studies have analyzed epigenetic marks, the enzymes mediating these marks, and the extent of their active contribution to CRC tumor development and progression [2, 3]. For instance, several methylationrelated enzymes have been found to be clinically relevant to CRC [15]. Among these, some of the most prominent histone methyltransferases (HMTs) that have been targeted for preclinical and clinical treatment of CRC are discussed in Section 4 of this chapter [15, 16]. On the other hand, a comparatively less number of histone demethylases (HDMs) have been validated as pertinent to CRC pathogenesis. As important regulators of colon cell transformation, histone deacetylases (HDAC) have also emerged as prominent markers of early carcinogenic events due to their unique role in maintaining higher-order chromatin structure [17].

In this chapter, we also highlight a few chemical inhibitors relevant to epigenetic therapy. However, we also noted that among the CRC-associated epigenetic enzymes, only a few of them have potent inhibitors available [15]. This suggests that the knowledge concerning targeting these enzymes for CRC is still insufficient and needs further evaluation. For example, only a few DNA methyltransferase (DNMT) and HMT inhibitors have been used in CRC cells [2], and a handful of Food and Drug Administration (FDA)-approved HDAC inhibitors are currently being explored for the treatment of solid tumors including CRC [18].

Unfortunately, the use of such epigenetic-based inhibitors has not been without limitations. Major drawbacks, such as adverse side effects and lack of clinical efficacy, have limited their use as single agents. Therefore, many inhibitors show more promise in combination therapy with chemotherapies suggesting that the full therapeutic potential of epigenetic therapy will perhaps be best realized in combination with other anti-cancer agents [19, 20]. This is also complemented by the recent understanding that there is a strong interplay between immune and cancer cells within the tumor microenvironment [21]. Recent studies in CRC cells have shown promising combinations of epigenetic and immunomodulatory drugs. By reversing expression changes of genes involved in immune suppression and thus enhancing expression of tumor-associated antigens, cancer cells potentially become more sensitive to immune checkpoint inhibitors [22]. These and other discoveries have established a highly promising basis for studies using combined epigenetic and immunotherapeutic agents for treating CRC.

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tumor suppressor *p21WAF*<sup>1</sup>

*Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer*

associated with a better prognosis for early-stage CRC patients [26].

Over the past decade, significant advances in our understanding of the CRC "epigenome" have revealed that most CRC cases harbor alterations in their histone modification states, particularly regarding aberrant histone methylation and acetylation [6, 15, 23]. Importantly, these abnormal histone marks are highly recurrent and have recently been used as biomarkers to predict the clinical outcome in CRC patients [2]. These include changes in the global patterns of specific histone modifications. For example, Tamagawa et al. showed that global changes in histone H3K4me2, H3K9ac, and H3K9me2 in metachronous liver metastasis correlated to overall survival of CRC patients [24]. Specifically, low H3K4me2 levels were shown to correlate with overall poor prognosis [24]. Likewise, other studies have identified reduced levels of H3K9me3 and H4K20me3 as diagnostic biomarkers for CRC in circulating nucleosomes which correlated with poor patient outcome [25]. Conversely, high H4K20me3 and H3K9me3, as well as low nuclear expression of H3K4me3, were

Furthermore, since reduction or enrichment of these marks frequently occurs at the promoters of key CRC-related oncogenes and tumor suppressors, this results in detrimental changes in gene expression that form the basis of tumorigenesis [15, 27]. For instance, H3K4me3, when found to be elevated in CRC primary tumors and cell lines, resulted in activated *Wingless-type* (*WNT*) signaling and target gene expression via interaction between *SET domain-containing protein 1A* (*SETD1A*) and *β-catenin* [28]. Meanwhile, another study revealed that low H3K4me1/2/3 levels were associated with hypoxia-induced silencing of *MLH1* in SW480 cells, which is a key event in the DNA mismatch defects linked to the development of sporadic CRC [29]. Yokoyama's group also demonstrated a role for the well-recognized repressive mark H3K9me3, revealing that its increased levels in metastatic CRC patient-derived cells correlated with enhanced cell motility [30]. Interestingly, this coincided with repression of *Ataxia-telangiectasia mutated* (*ATM*) and *p53-associated KZNF protein* (*APAK*), leading to a defect in p53-dependent apoptosis [30]. Moreover, enrichment of another repressive mark, H3K27me3, was associated with poor CRC patient prognosis while elevated H3K79me2 was shown to enhance interleukin (IL)-22-induced stemness in CRC cells [31, 32]. Intriguingly, more recent studies have also shown that mutations in specific methylation sites could promote CRC development. For instance, the Shah and Lu groups identified histone 3 lysine 36-to-methionine (H3K36M) substitution mutations in CRC patient samples, which promoted more undifferentiated sarcomas *in vivo* [33, 34]. This suggests that H3K36 methylation potentially constitutes a major tumor suppressive epigenetic mark. In addition to abnormal methylation, disruption of histone acetylation patterns also contributes to CRC pathogenesis, particularly relating to transcriptional inactivation of tumor suppressor genes and, sometimes, activation of oncogenes. For example, Richon et al. showed that hypoacetylation at the promoter of the

led to its repression, an effect that was reversed by

inhibition of HDAC activity [35]. Conversely, mass spectrometry-based analyses used to quantify global alterations of histone modifications in CRC samples identified H3K27ac as a modification frequently upregulated in CRC [36]. In fact, one study highlighted the effects of aspirin in reducing the enrichment of H3K27ac in the promoters of *inducible nitric oxide synthase* (*iNOS*), *tumor necrosis factor alpha* (*TNF-α*), and *IL-6* [37]. This in turn corresponded to the dramatic reduction of the mRNA and protein levels of these genes, which suppressed inflammatory colitis symptoms and CRC tumor burden [37]. Taken together, these studies emphasize

*DOI: http://dx.doi.org/10.5772/intechopen.82572*

**2. Epigenetic modifications in CRC**

**2.1 Histone modifications**
