**4. The role of major epigenetic enzymes in CRC and therapeutic strategies for targeting them**

### **4.1 Histone methyltransferases and demethylases**

As discussed so far, aberrant changes in epigenetic modifications can significantly contribute to CRC progression. It is therefore unsurprising that many of the epigenetic enzymes mediating these modifications are themselves deregulated during the initiation and progression of CRC. Here, we describe the significance of changes in the expression levels of two such families of enzymes that oppose each other in terms of function, namely histone methyltransferases (HMTs) and demethylases (HDMs). Although changes in the expression or activity levels of several methylation-related enzymes have been linked to CRC, in most cases only a limited knowledge regarding the molecular mechanisms by which these enzymes contribute to disease development exists [15]. We summarize current knowledge regarding some of the preclinical validated implications of these enzymes as proof of principle for the employment of epigenetic agents in CRC. We also briefly discuss potential mechanisms of action of these enzymes as well as the advantages of targeting them using combinatorial over monotherapy approaches.

Histone lysine methyltransferases (HKMTs) have been widely studied across multiple solid tumor types including CRC [97]. For instance, studies in a preclinical model of CRC found that increased expression and activity of *SET and MYND domain containing 3* (*SMYD3*), a well-known HKMT, was strongly correlated with tumorigenesis. Moreover, RNAi-mediated depletion of *SMYD3* significantly impaired CRC cell proliferation, indicating a crucial role of *SMYD3* in maintaining CRC malignancy [98]. More recent studies suggest a putative mechanism by which this overexpression might occur by demonstrating that hypomethylation of the *SMYD3* promoter was observed in CRC tumor tissues compared to adjacent normal tissues. Further subgroup clinicopathological analyses showed that this hypomethylation was observed with stage III and IV tumors as defined by moderate to well-differentiated histology and positive lymph node metastasis [99].

Another well-studied HKMT, *enhancer of zeste 2* (*EZH2*), is also frequently deregulated in CRC. Both mRNA and protein levels of *EZH2* were found to be significantly increased in CRC tissues compared to non-cancerous counterparts [16]. Additionally, increased *EZH2* expression was directly correlated with tumor size, metastases, and overall worse disease-free survival of CRC patients [100]. He et al. also showed that siRNA-mediated depletion of *EZH2* inhibited the proliferation and migration of SW620 CRC cells, while inducing apoptosis and G0/G1 cell cycle arrest [101]. Another mechanistic study also revealed that knockdown of *EZH2* significantly reduced CRC cell invasion and *matrix metalloproteinases 2*/*9* (*MMP2*/*9*) secretion *in vitro* while promoting increased overall survival and decreased lung metastasis *in vivo* [102]. Furthermore, this *EZH2*-induced CRC cell invasion was mediated by direct binding of *Signal transducer and activator of transcription 3*

**89**

*Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer*

predictor of poor survival outcome in CRC patients [32].

(*STAT3*) to the *EZH2* promoter, resulting in downregulation of the vitamin D receptor (VDR) [102]. Interestingly, an association between a missense variant in *EZH2* and risk of CRC was discovered by the Li group. They identified that the presence of the rs2302427 variant showed a significant association with increased CRC susceptibility [103]. Recent studies point to other mechanistic roles of HKMTs in CRC. For example, depletion of *SETD1A*, a member of the trithorax (TrxG) family of HMTs, inhibited CRC cell growth and colony formation in part by decreasing expression of approximately 50% of *Wnt*/*β-catenin* target genes [28]. Finally, in a mouse model, IL-22-mediated activation of *disruptor of telomeric silencing 1-like* (*DOT1L*) promoted CRC stemness and tumorigenic potential and was considered a

Protein arginine methyltransferases (PRMTs), although studied to a lesser extent, have also been shown to play critical roles in CRC malignancy via activation of *Wnt*/*β-catenin* and NF-ĸB signaling [104]. *CARM1*, for example, is an important positive modulator of *Wnt*/*β-catenin* transcription and was found to promote survival and anchorage-independent growth of CRC cells with aberrantly activated *Wnt*/*β-catenin* signaling [105]. Meanwhile, our lab and others have shown that *protein arginine methyltransferase 5* (*PRMT5*) was overexpressed in CRC cells and patient-derived primary tumors, which correlated with increased cell growth, migration, invasion, and NF-ĸB activation as well decreased overall patient survival [106–109]. The enzymes catalyzing removal of methylation marks, HDMs, are perhaps the least studied among the enzymes mentioned thus far and only a few have been implicated as playing tumor suppressive or oncogenic roles in CRC. *LSD1*, *KDM4B*, *KDM4C*, and *KDM5B* have all been shown to play pro-tumorigenic roles by promoting CRC cell growth and metastasis, whereas HDMs, such as *JMJD3* and *JMJD1B*, have been implicated as tumor suppressors [15]. Taken together, these data provide strong support for the continued development of selective and potent small-molecule inhibitors against these methylation-modifying enzymes as promis-

Disruption of epigenetic regulation in CRC mediated by deregulated HMTs, DNMTs, and HDMs has garnered increasing interest in recent years. In this section, we aim to review the current status on the development of therapeutic strategies to modulate histone methylation for CRC treatment. The current therapeutics including pre-clinical and clinical agents that target epigenetic enzymes in CRC are listed in **Table 1**. Thus far, more than 20 histone-methylation enzymes have been found to be clinically relevant to CRC, including 17 oncoproteins and 8 tumor suppressors, although their exact mechanisms of action are not fully understood [15]. Furthermore, more than 20 small-molecule inhibitors targeting HMTs, DMNTs, and HDMs have been employed for preclinical or clinical studies. For example, treatment of DLD1 colon cell line and primary CRC cells with a potent HKMT inhibitor EPZ004777 (anti-*DOT1L*) resulted in significant reduction in sphere formation *in vitro*, thus inhibiting cell growth [32]. Other HKMT inhibitors, such as BCI-121 and Chaetocin, have significantly suppressed CRC cell growth and migration by inhibiting *SMYD3* and *SUV39H1*, respectively [98]. Notably, inhibitors against the HKMT *EZH2* have yielded some of the most promising results for treating CRC. DZNep, an indirect *EZH2* inhibitor, induced apoptosis in CRC cell lines and stem cells, while GSK346 impaired the migratory potential of CRC cells

and reduced H3K27me3 levels in Colo205 and HT-29 cells (**Table 1**) [110].

gained prominence in the cancer field, and only a couple of these have made it

Unlike HKMTs, the development of inhibitors against PRMTs has only recently

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

ing therapeutic agents for CRC.

**4.2 Targeting HMTs and HDMs in CRC**

*Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.82572*

(*STAT3*) to the *EZH2* promoter, resulting in downregulation of the vitamin D receptor (VDR) [102]. Interestingly, an association between a missense variant in *EZH2* and risk of CRC was discovered by the Li group. They identified that the presence of the rs2302427 variant showed a significant association with increased CRC susceptibility [103]. Recent studies point to other mechanistic roles of HKMTs in CRC. For example, depletion of *SETD1A*, a member of the trithorax (TrxG) family of HMTs, inhibited CRC cell growth and colony formation in part by decreasing expression of approximately 50% of *Wnt*/*β-catenin* target genes [28]. Finally, in a mouse model, IL-22-mediated activation of *disruptor of telomeric silencing 1-like* (*DOT1L*) promoted CRC stemness and tumorigenic potential and was considered a predictor of poor survival outcome in CRC patients [32].

Protein arginine methyltransferases (PRMTs), although studied to a lesser extent, have also been shown to play critical roles in CRC malignancy via activation of *Wnt*/*β-catenin* and NF-ĸB signaling [104]. *CARM1*, for example, is an important positive modulator of *Wnt*/*β-catenin* transcription and was found to promote survival and anchorage-independent growth of CRC cells with aberrantly activated *Wnt*/*β-catenin* signaling [105]. Meanwhile, our lab and others have shown that *protein arginine methyltransferase 5* (*PRMT5*) was overexpressed in CRC cells and patient-derived primary tumors, which correlated with increased cell growth, migration, invasion, and NF-ĸB activation as well decreased overall patient survival [106–109]. The enzymes catalyzing removal of methylation marks, HDMs, are perhaps the least studied among the enzymes mentioned thus far and only a few have been implicated as playing tumor suppressive or oncogenic roles in CRC. *LSD1*, *KDM4B*, *KDM4C*, and *KDM5B* have all been shown to play pro-tumorigenic roles by promoting CRC cell growth and metastasis, whereas HDMs, such as *JMJD3* and *JMJD1B*, have been implicated as tumor suppressors [15]. Taken together, these data provide strong support for the continued development of selective and potent small-molecule inhibitors against these methylation-modifying enzymes as promising therapeutic agents for CRC.

## **4.2 Targeting HMTs and HDMs in CRC**

Disruption of epigenetic regulation in CRC mediated by deregulated HMTs, DNMTs, and HDMs has garnered increasing interest in recent years. In this section, we aim to review the current status on the development of therapeutic strategies to modulate histone methylation for CRC treatment. The current therapeutics including pre-clinical and clinical agents that target epigenetic enzymes in CRC are listed in **Table 1**. Thus far, more than 20 histone-methylation enzymes have been found to be clinically relevant to CRC, including 17 oncoproteins and 8 tumor suppressors, although their exact mechanisms of action are not fully understood [15]. Furthermore, more than 20 small-molecule inhibitors targeting HMTs, DMNTs, and HDMs have been employed for preclinical or clinical studies. For example, treatment of DLD1 colon cell line and primary CRC cells with a potent HKMT inhibitor EPZ004777 (anti-*DOT1L*) resulted in significant reduction in sphere formation *in vitro*, thus inhibiting cell growth [32]. Other HKMT inhibitors, such as BCI-121 and Chaetocin, have significantly suppressed CRC cell growth and migration by inhibiting *SMYD3* and *SUV39H1*, respectively [98]. Notably, inhibitors against the HKMT *EZH2* have yielded some of the most promising results for treating CRC. DZNep, an indirect *EZH2* inhibitor, induced apoptosis in CRC cell lines and stem cells, while GSK346 impaired the migratory potential of CRC cells and reduced H3K27me3 levels in Colo205 and HT-29 cells (**Table 1**) [110].

Unlike HKMTs, the development of inhibitors against PRMTs has only recently gained prominence in the cancer field, and only a couple of these have made it

*Advances in the Molecular Understanding of Colorectal Cancer*

**strategies for targeting them**

**4.1 Histone methyltransferases and demethylases**

It is also noteworthy that many CRC tumors demonstrate mixed characteristics compatible with two or more of these subtypes, which may represent a transition phenotype or intratumoral heterogeneity, while others cannot be precisely classified into any of these pre-defined subgroups [95]. Furthermore, these classifications often lack incorporation of the molecular markers used for traditional TNM staging of CRC [96]. Taken together, these challenges as well as the existing incongruity between the various systems illustrate the need to further refine these consensus classifications by developing more progressive and integrated approaches.

**4. The role of major epigenetic enzymes in CRC and therapeutic** 

targeting them using combinatorial over monotherapy approaches.

to well-differentiated histology and positive lymph node metastasis [99].

Another well-studied HKMT, *enhancer of zeste 2* (*EZH2*), is also frequently deregulated in CRC. Both mRNA and protein levels of *EZH2* were found to be significantly increased in CRC tissues compared to non-cancerous counterparts [16]. Additionally, increased *EZH2* expression was directly correlated with tumor size, metastases, and overall worse disease-free survival of CRC patients [100]. He et al. also showed that siRNA-mediated depletion of *EZH2* inhibited the proliferation and migration of SW620 CRC cells, while inducing apoptosis and G0/G1 cell cycle arrest [101]. Another mechanistic study also revealed that knockdown of *EZH2* significantly reduced CRC cell invasion and *matrix metalloproteinases 2*/*9* (*MMP2*/*9*) secretion *in vitro* while promoting increased overall survival and decreased lung metastasis *in vivo* [102]. Furthermore, this *EZH2*-induced CRC cell invasion was mediated by direct binding of *Signal transducer and activator of transcription 3*

As discussed so far, aberrant changes in epigenetic modifications can significantly contribute to CRC progression. It is therefore unsurprising that many of the epigenetic enzymes mediating these modifications are themselves deregulated during the initiation and progression of CRC. Here, we describe the significance of changes in the expression levels of two such families of enzymes that oppose each other in terms of function, namely histone methyltransferases (HMTs) and demethylases (HDMs). Although changes in the expression or activity levels of several methylation-related enzymes have been linked to CRC, in most cases only a limited knowledge regarding the molecular mechanisms by which these enzymes contribute to disease development exists [15]. We summarize current knowledge regarding some of the preclinical validated implications of these enzymes as proof of principle for the employment of epigenetic agents in CRC. We also briefly discuss potential mechanisms of action of these enzymes as well as the advantages of

Histone lysine methyltransferases (HKMTs) have been widely studied across multiple solid tumor types including CRC [97]. For instance, studies in a preclinical model of CRC found that increased expression and activity of *SET and MYND domain containing 3* (*SMYD3*), a well-known HKMT, was strongly correlated with tumorigenesis. Moreover, RNAi-mediated depletion of *SMYD3* significantly impaired CRC cell proliferation, indicating a crucial role of *SMYD3* in maintaining CRC malignancy [98]. More recent studies suggest a putative mechanism by which this overexpression might occur by demonstrating that hypomethylation of the *SMYD3* promoter was observed in CRC tumor tissues compared to adjacent normal tissues. Further subgroup clinicopathological analyses showed that this hypomethylation was observed with stage III and IV tumors as defined by moderate

**88**


#### **Table 1.**

*Overview of pre-clinical and clinical drugs that target epigenetic enzymes in CRC.*

to the clinical trial phase thus far. AMI-1, which inhibits *PRMT1* and *PRMT5*, demonstrated antiproliferative activity in CRC cells and xenograft mouse models [106]. However, further *in vivo* validation studies are needed, and it has not

**91**

*Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer*

potential for synergy between the two classes of HDM inhibitors.

entered clinical trial yet. Another promising *PRMT5* inhibitor that recently made it to Phase I clinical trials is GSK3326595, which potently inhibited tumor growth *in vitro* and *in vivo* [111]. Trials with GSK3326595 are currently being conducted in adult subjects with relapsed and/or refractory solid tumors (NCT02783300). Additionally, inhibitors targeting HDMs are even fewer in number and have shown limited efficacy in suppressing CRC cell growth. For example, *KDM4A*/*C* inhibitors were ineffective in blocking HCT116 CRC cell growth when used in isolation [112]. However, they exhibited potent antiproliferative effects in combination with another HDM inhibitor, NCL-2, which targets *LSD1* [113]. These data suggest a

Finally, the use of DNMT inhibitors for CRC treatment has also shown some exciting promise. In studies using CRC cell lines, suppression of *DNMT1* and *DNMT3B* resulted in significant reduction in methylation, which correlated with the re-expression of tumor suppressor genes. This also resulted in induction of apoptosis as well as reduced cell proliferation and stemness [114]. Notably, studies with the *DNMT1* inhibitor, 5-aza-2′-deoxycytidine (decitabine), exhibited its ability to re-sensitize colorectal tumors to both irinotecan and 5-FU, thus becoming a major component of the treatment regimen for CRC in the clinic [19]. Another recent preclinical study showed that combination of the anti-*EGFR* inhibitor, gefitinib and decitabine showed highly synergistic inhibition of CRC cell proliferation and migration [115]. Additional combination regimens are outlined in **Table 1**.

Acetylation of histones by acetyltransferases (HATs) and removal of these acetyl marks by HDACs are essential events for the maintenance of normal chromatin organization and function [116]. However, as is often the case in cancer, these enzymes are dysregulated, leading to increased chromosomal instability and aberrant gene expression changes [117]. To date, only a handful of HATs have been reported as contributing to the pathogenesis of CRC. Here, we describe the role of a few of these HATs namely *p300*/*CREB-binding protein* (*p300*/*CBP*), *GCN5*, *N-Acetyltransferase 10* (*Nat10*), and *Human males absent on the first* (*hMOF*). Assessment of 262 CRC samples from patients receiving 5-FU treatment demonstrated that low expression of *p300*/*CBP* in CRC tissue was closely associated with poor clinical response to 5-FU based-chemotherapy [118]. Furthermore, low *p300*/*CBP* expression also correlated with poor disease-free survival and increased early disease progression in the same patients [118]. Mechanistic studies also uncovered that 5-FU induced degradation of *p300*/*CBP* which was dependent on chaperone-mediated autophagy involving *heat-shock cognate protein 70 kDa* (*hsc70*) and *lysosomal-associated membrane protein 2A* (*LAMP2A*). In short, degradation of *p300*/*CBP* was found to be relevant to chemoresistance to 5-FU, since blocking this

degradation also enhanced 5-FU's cytotoxicity in CRC cells [118].

Conversely, another HAT *GCN5* has been implicated in promoting CRC cell growth via its upregulation rather than downregulation. One study found that *GCN5* overexpression in human colon adenocarcinoma tissues was attributed to the activities of the transcription factors, *c-Myc* and *E2F transcription factor 1* (*E2F1*) [119]. Depletion of *c-Myc* inhibited CRC cell proliferation mainly by downregulating GCN5 transcription, an effect that was rescued by ectopic expression of *GCN*5. However, ectopic overexpression of *E2F1* had the opposite effect by suppressing *GCN5* levels, thus inducing cell death. Furthermore, inhibition of *GCN5* with CPTH2, a HAT inhibitor, also suppressed CRC cell growth, revealing an avenue of great therapeutic potential [119]. Other HATs implicated in CRC include *Nat10* and *hMOF*, which were downregulated in CRC tissues. Particularly, recent studies

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

**4.3 Acetyltransferases and deacetylases**

### *Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer DOI: http://dx.doi.org/10.5772/intechopen.82572*

entered clinical trial yet. Another promising *PRMT5* inhibitor that recently made it to Phase I clinical trials is GSK3326595, which potently inhibited tumor growth *in vitro* and *in vivo* [111]. Trials with GSK3326595 are currently being conducted in adult subjects with relapsed and/or refractory solid tumors (NCT02783300). Additionally, inhibitors targeting HDMs are even fewer in number and have shown limited efficacy in suppressing CRC cell growth. For example, *KDM4A*/*C* inhibitors were ineffective in blocking HCT116 CRC cell growth when used in isolation [112]. However, they exhibited potent antiproliferative effects in combination with another HDM inhibitor, NCL-2, which targets *LSD1* [113]. These data suggest a potential for synergy between the two classes of HDM inhibitors.

Finally, the use of DNMT inhibitors for CRC treatment has also shown some exciting promise. In studies using CRC cell lines, suppression of *DNMT1* and *DNMT3B* resulted in significant reduction in methylation, which correlated with the re-expression of tumor suppressor genes. This also resulted in induction of apoptosis as well as reduced cell proliferation and stemness [114]. Notably, studies with the *DNMT1* inhibitor, 5-aza-2′-deoxycytidine (decitabine), exhibited its ability to re-sensitize colorectal tumors to both irinotecan and 5-FU, thus becoming a major component of the treatment regimen for CRC in the clinic [19]. Another recent preclinical study showed that combination of the anti-*EGFR* inhibitor, gefitinib and decitabine showed highly synergistic inhibition of CRC cell proliferation and migration [115]. Additional combination regimens are outlined in **Table 1**.
