**2.3 Noncoding RNAs**

*Advances in the Molecular Understanding of Colorectal Cancer*

CRC development and progression.

**2.2 DNA methylation**

described in Section 1 [39].

the differential abundance of key repressive and activating histone methylation and acetylation marks in CRC and suggests their role in regulating genes associated with

DNA methylation constitutes the first recognized epigenetic alteration in CRC [38]. Usually, global DNA hypomethylation is frequently seen, which occurs gradually and early in the process of CRC carcinogenesis [38]. More precisely, global DNA hypomethylation mainly takes place on cytosine guanine (CpG) dinucleotides within pericentromeric regions. Initially, this hypomethylation in CRC was hypothesized to be associated mainly with widespread oncogene activation but has now been linked predominantly to increased genomic instability [3]. This increased accumulation of chromosome breakage and overall chromosomal instability contributes to a prevalent subtype of CRCs known as the CIN phenotype as we briefly

Notably, hypomethylation typically occurs in concert with systematic and discrete DNA hypermethylation events at the promoters of genes involved in DNA repair, apoptosis, proliferation, angiogenesis, adhesion, and invasion [38, 40]. DNA hypermethylation is the most extensively characterized epigenetic alteration in CRC, occurring at CpG dinucleotide-dense regions, called CpG islands, which are present in about 60% of genes [6, 41]. Apart from CpG islands, DNA hypermethylation has also been extensively observed within the first exonic/intronic regions of some genes and generally results in transcriptional silencing [42]. Some of the most frequently hypermethylated genes in CRC include *Adenomatous polyposis coli* (*APC*), *Cyclin-dependent kinase inhibitor 2A* (*p16INK4a*/*CDKN2A*), *Tissue inhibitor of metalloproteinases 3* (*TIMP3*), *O-6-Methylguanine-DNA methyltransferase* (*MGMT*), *Secreted frizzled related protein 1* (*SFRP1*), *Transmembrane protein with epidermal growth factor* (*EGF*) *like and two follistatin like domains 2* (*TMEFF2*), *Heparan sulfate-glucosamine 3-sulfotransferase 2* (*HS3ST2*/*3OST2*), *Ras association domain family member 1* (*RASSF1A*), and *GATA binding protein 4* (*GATA4*) [43].

Another subtype of CRCs with extensive patterns of promoter methylation, known as the CIMP phenotype as described in Section 1, is also characterized by aberrant DNA methylation at genes with roles in CRC initiation or progression [44]. For instance, using a qPCR-based technique, one group identified genes with the highest percentage of methylation in CRC patients including *Runt related transcription factor 3* (*RUNX3*), *Protocadherin 10* (*PCDH10*), *Secreted frizzled related protein 5* (*SFRP5*), *Insulin-like growth factor 2* (*IGF2*), and *Hepatocyte nuclear factor 1 homeobox A* (*HNF1*) *homeobox B* (*Hnf1b*) [45]. Moreover, these genes were observed to have the most promising biomarker potential because of the frequent gene repression patterns [45]. Other commonly hypermethylated genes, such as *Sex-determining region Y* (*SRY*)*-related HMG-box* (*SOX17*) and *Apoptosis-associated speck-like protein containing a CARD* (*ASC*)/*target of methylation-induced silencing* (*TMS1*), were differentially methylated based on the staging of the disease [46–48], whereas *MLH1*, *p16*, *Death-associated protein kinase 1* (*DAP-kinase*), *Ras association domain family member 2* (*RASSF2A*), and *WNT inhibitory factor 1* (*Wif-1*) were

In summary, these data strongly support the promising utility of DNA methylation as a critical diagnostic marker for CRC. Unfortunately, this has not necessarily translated into their prognostic or predictive use in clinical practice [50]. This can be attributed to significant variability in sensitivity, specificity, and reproducibility between diverse patient cohorts and gene expression platforms, which ultimately impacts the prognostic value of many tests. Currently, two FDA-approved

regarded as plasma or serum detection markers for CRC [49].

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Another epigenetic regulatory mechanism frequently deregulated in CRC involves the role of noncoding RNA (ncRNAs). Specifically, aberrations of microR-NAs (miRNAs) expression, a major class of ncRNAs, are often observed in CRC and are considered to play a major role in tumorigenesis and CRC progression [23, 53]. These observations are consistent with the fact that miRNAs tend to exert oncogenic or tumor-suppressive effects. For example, miRNAs, such as miR-141, miR-200c, miR-145, miR-373, miR-520c, miR-135a, and miR-135b, have all been shown to affect CRC by regulating epithelial differentiation, WNT signaling, and CRC cell migratory and invasive potential [54]. Other miRNAs implicated in CRC include miRNA-124a and miRNA-34b/34c, which were shown to regulate the cell cycle and TP53 pathway, respectively [55]. Several miRNAs are also associated with epithelialto-mesenchymal transition (EMT) in CRC. miR-15/16, miR-140, and miR-200 family members were shown to be associated with suppression of EMT and tumor cell metastatic potential while miR-21 enhanced this process [56].

Other studies have highlighted that alterations in the expression pattern of miRNAs in CRC were considered diagnostic, prognostic, or chemosensitivity markers [57]. For instance, high levels of miR-320 and miR-498 were correlated with progressionfree survival in stage II CRC [58], while miR-21 abundance was associated with poor patient response to 5-fluorouracil adjuvant chemotherapy [59]. On the other hand, induced suppression of miR-21 promoted the sensitization of CRC cells to chemotherapeutic regimens [60, 61]. Another study by Toiyama et al. demonstrated a correlation between elevated serum miR-200c levels and stage IV CRC compared to earlier stages. Furthermore, high serum miR-200c showed a significantly positive correlation with lymph node metastasis, distant metastasis, and prognosis [62]. A comprehensive look of miRNAs as CRC biomarkers is reviewed by several other sources [60].

There is also ample evidence of miRNAs being downregulated in CRC and thus playing tumor-suppressive roles [63]. Arndt et al. showed that reduced levels of miR-133a as well as enrichment of miR-224 were associated with CRC initiation [64]. Moreover, this study and others also revealed that CRC patients at the adenomatous and carcinoma stages consistently exhibited reduced steady-state levels of miR-143 and miR-145 [63, 64]. Another classic example includes the miR-34 family. Transfection of miR-34a into CRC cells led to induction of apoptosis and inhibition of cell proliferation in part by amplifying the p53-mediated apoptotic response [65, 66]. Intriguingly, p53 has been shown to regulate miR-34a, suggesting a positive feedback loop between the two in which miR-34a could partly mediate the tumor-suppressive roles of p53 [55].

Interestingly, miRNA deregulation can also induce aberrant activity of many of the components of the epigenetic machinery [67]. Take *DNMT3A* for example, which has been identified as a miR-143 target and is associated with CRC via downregulation of miR-143 and subsequent increase in *DNMT3A* expression levels [68]. Other examples include miR-140 and miR-449, which have been shown to target and downregulate HDAC1 and 4, respectively, thus exerting their tumor-suppressive effects [69, 70]. Taken together, these findings underscore the importance of miRNAs in exerting both oncogenic and antitumor roles in CRC, which may serve as the basis for the development of novel prognostic and therapeutic markers.
