Epigenetic Alterations Upstream and Downstream of p53 Signaling in Colorectal Carcinoma

Simple Summary Colorectal cancer (CRC) belongs to the most common cancer types. It is well known that half of all CRC possess missense mutations in the TP53 tumor suppressor gene. However, the entire signaling cascade upstream and downstream of the p53 protein may also contribute to CRC development, if relevant players in this signaling cascade lost their function. Besides p53 loss-of-function by mutations, epigenetic changes (DNA methylation, post translational modifications of histones, micro-RNAs) play a vital role in CRC development. In the present review, we concentrated on the epigenetic modifications related to the entire p53 signal transduction cascade upstream and downstream of p53. Indeed, numerous epigenetic aberrations influence the tumor suppressor function of p53 independent of missense mutations. Thus, the role of p53 for CRC development, therapy response and survival prognosis of patients may be much more complex than predicted earlier. Hence, we are in need to use novel diagnostic methods that are capable of evaluating the genetic and epigenetic changes in the “p53 signalome”, so that diagnosis and management of CRC will improve. Abstract Colorectal cancer (CRC) belongs to the most common tumor types, and half of all CRC harbor missense mutations in the TP53 tumor suppressor gene. In addition to genetically caused loss of function of p53, epigenetic alterations (DNA methylation, histone modifications, micro-RNAs) contribute to CRC development. In this review, we focused on epigenetic alterations related to the entire p53 signaling pathway upstream and downstream of p53. Methylation of genes which activate p53 function has been reported, and methylation of APC and MGMT was associated with increased mutation rates of TP53. The micro-RNA 34a activates TP53 and was methylated in CRC. Proteins that regulate TP53 DNA methylation, mutations, and acetylation of TP53-related histones were methylated in CRC. P53 regulates the activity of numerous downstream proteins. Even if TP53 is not mutated, the function of wildtype p53 may be compromised if corresponding downstream genes are epigenetically inactivated. Thus, the role of p53 for CRC development, therapy response, and survival prognosis of patients may be much more eminent than previously estimated. Therefore, we propose that novel diagnostic devices measuring the entirety of genetic and epigenetic changes in the “p53 signalome” have the potential to improve the predictive and prognostic power in CRC diagnostics and management.


Introduction
Since the early 19th century and the ground-breaking experiments of Gregor Mendel, genetics is fundamental in biology in general and also specifically in cancer biology [1]. Epigenetics emerged as new field, which complements many genetic mechanisms in evolution of life on earth, organismic homeostasis, and pathophysiology of diseases as well.
Mutations in DNA mismatch repair genes do not only predispose to mutations in driver genes, but also to another characteristic feature of colorectal cancer, i.e., microsatellite instability (MSI) [24]. Because of impaired DNA mismatch repair, there is a predisposition for DNA mutations, and three phenotypes can be distinguished: MSI stable, MSI low, and MSI high, the latter one with a better prognosis than the others [25]. Epigenetic inactivation of other DNA repair genes contributes to mutations of driver genes in CRC (e.g., the mismatch repair genes hMLH1, hMSH2, and the gene coding for O 6 -methyguanine-DNA methyltransferase, MGMT) [26].
Interestingly, epigenetic alterations occur more frequently in colorectal cancer than gene mutations. A large number of hyper-and hypomethylations appear in genes and miRNAs of a majority of colorectal cancers [27]. It is estimated that 600-800 genes are transcriptionally silenced by CpG island methylation and that miRNAs also considerably affect transcriptional repression in this tumor entity [28,29]. The list of genes belonging to this CpG island hypermethylated phenotype (CIMP) is still growing and this phenotype is of clinical prognostic significance [30][31][32]. Several different epigenotypes have been defined in colorectal cancer with different prognostic outcome (high and low methylator phenotypes associated with specific high and low MSI and mutational profiles in driver genes [33,34]).
Even if CIMP epigenotypes can be identified consisting of different genetic and epigenetic profiles, there are numerous specific interactions between single genes (e.g., TP53) and aberrant methylation in p53-related genes.
A lot of attention has been paid on mutations in the TP53 gene and the related loss of function. Rather than genetic alterations, we focus in the present paper on epigenetic changes in the TP53 gene and also on epigenetics in the signaling cascade upstream and downstream of the p53 protein. If we assume that epigenetic changes in genes upstream and downstream of p53 also contribute to alterations in p53 signaling, the oncogenic potential of the entire epigenetically silenced p53 signaling pathways in addition to 50-60% mutations in TP53 itself is much larger than estimated yet. Therefore, we performed a systematic review on the interactions between p53 and methylation changes in genes upstream of the p53 signaling cascade or downstream genes, which are regulated by p53. Our overview is based on a PubMed search with the keywords "colorectal + epigenetic + p53" and "colorectal + methylator phenotype + p53" as of 19 May 2021.

Epigenetic Alterations Upstream of p53 2.1. Methylation Status of p53-Activating Genes
A number of genes mostly acting as tumor suppressors or oncogenes regulate the activity of p53 (Table 1, Figure 1). Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a tumor suppressor protein, which stabilizes p53 expression. Its demethylation causes growth inhibition by G2/M cell cycle arrest and apoptosis induction. UCHL1 methylation has been reported in 22/31 CRC cases (=71%) [35].
Ras-association domain family member 10 (RASSF10) is a RAS-associated domain family member, which activates p53 signaling and sensitizes to anti-cancer drugs (e.g., docetaxel). Demethylation of RASSF10 induced apoptosis and inhibited proliferation in 54/89 CRC cases (=61%). The RASSF10 methylation status was positively associated with tumor stage and metastasis [37].
Ras-association domain family member 10 (RASSF10) is a RAS-associated domain family member, which activates p53 signaling and sensitizes to anti-cancer drugs (e.g., docetaxel). Demethylation of RASSF10 induced apoptosis and inhibited proliferation in 54/89 CRC cases (=61%). The RASSF10 methylation status was positively associated with tumor stage and metastasis [37].
Heparanase 2 (HPSE2) acts as tumor suppressor, which regulates p53 signaling leading to G1 arrest of the cell cycle. Hypermethylation of HPSE2 significantly correlated with shorter survival times of CRC patients [38].
The proto-oncogene BMI1 (B lymphoma Mo-MLV insertion region 1 homologue) is an epigenetic repressor by remodeling chromatin. P14 ARF and wildtype p53 were upregulated in BMI1-mutated CRC cases [39]. P14 ARF is required for the induction of p53 expression and apoptosis.
The protein stability of p53 is regulated by p14 ARF , which is also designated as cyclindependent kinase inhibitor 2A (CDKN2A). This protein interacts with MDM2 (mouse double minute 2 homologue) and thereby antagonizes the MDM2-dependent degradation of p53. Demethylation of the CDKN2A promoter was associated with MDM2 expression, while CDKN2A hypermethylation resulted in cytosolic translocation and inactivation of The proto-oncogene BMI1 (B lymphoma Mo-MLV insertion region 1 homologue) is an epigenetic repressor by remodeling chromatin. P14 ARF and wildtype p53 were upregulated in BMI1-mutated CRC cases [39]. P14 ARF is required for the induction of p53 expression and apoptosis.
The protein stability of p53 is regulated by p14 ARF , which is also designated as cyclindependent kinase inhibitor 2A (CDKN2A). This protein interacts with MDM2 (mouse double minute 2 homologue) and thereby antagonizes the MDM2-dependent degradation of p53. Demethylation of the CDKN2A promoter was associated with MDM2 expression, while CDKN2A hypermethylation resulted in cytosolic translocation and inactivation of MDM2. Thereby, CDKN2A hypermethylation abrogated the activity of wildtype p53. CDKN2A hypermethylation was more frequently found in tumors with wildtype p53 than in those with mutated p53 [40]. CDKN2A demethylation was associated with nuclear (i.e., active) MDM2 expression in CRC [41]. In CRC, CDKN2A promoter hypermethylation was significantly correlated with p53 overexpression and MDM2 overexpression [42].

Methylation Status of p53-Inhibiting Genes
In addition to genes that activate p53, epigenetic regulation of genes, whose gene products inhibit p53, has also been reported ( Table 1).
O 6 -Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein whose downregulation by promoter hypermethylation predisposes genes to p53 mutations. MGMT promoter hypermethylation was significantly correlated to G > A transition mutations in the TP53 gene in 314 CRC patient samples [45]. This result was confirmed by other authors [46]. However, there was no association between MGMT methylation and TP53 mutations in 261 CRC biopsies from Afro-American patients, indicating that population-based differences may exist [47].
Deficiency of the tumor suppressor pentraxin 3 (PTX3) increases susceptibility to carcinogenesis by increasing DNA damage, p53 mutations and inactivation of p53 downstream signaling (MDM2, BAX, and CDKN1A/p21). Increasing rates of PTX3 promoter methylation were observed from normal colon epithelium to adenomas and CRC biopsies [48].
Trimethylated histone H3K27 binding in the PCAF (P300/CBP-associated factor) promoter attenuated transcription of this gene. Decreased PCAF impaired the acetylation of p53 and attenuated the p53-dependent transcription of p21, which resulted in increased cyclin D1 expression and Retinoblastoma 1 (RB1) phosphorylation as well as increased resistance to 5-fluorouracil [49].
Lactamase β (LACTB) is a tumor suppressor, which binds to the C-terminus of p53 to inhibit MDM2-mediated p53 degradation. LACTB was significantly downregulated in CRC due to promoter methylation [50].

Methylation Status of Other Genes
Epigenetic alterations have been associated with p53 without explicitly describing the direct causative relationship to p53 function (Tables 1 and 2).
Cyclooxygenase 2 (COX2) is a proinflammatory enzyme, which is involved in the progression of CRC. Methylation of the COX2 gene was found in 12/93 CRC biopsies (=13%) and 7/50 colorectal adenomas (=14%). COX2 methylation was inversely related to TP53 mutations, albeit the functional relevance of these mutations is not clear yet [52].

Acetylation Status of p53-Inhibiting Genes
LACTB was not only downregulated in CRC due to promoter methylation, but also due to histone deacetylation [50] (Tables 1 and 2).

P53 Regulation by Micro-RNAs
Micro-RNAs are involved in the regulation of p53 and its network at multiple levels [57] (Tables 1 and 2). This can occur by direct p53 targeting or indirectly by targeting p53 regulators (e.g., MDM2 and MDM4). Vice versa, p53 is a transcriptional regulator of numerous miRNAs, which contributes to its tumor suppressive function.
In tumor cells, including CRC, miRNAs can function in a tumor-suppressive (protective) or tumor-promoting (oncogenic) manner. MiR-339-5p was frequently downregulated and associated with poor patients' prognosis; miR-339-5p and miR1827 directly repressed MDM2 expression through binding to MDM2 3 -UTR, which elevated p53 protein expression and p53-mediated apoptosis and senescence. In parallel, it also inhibited migration, invasion, and the growth of CRC xenografts [58]. Other tumor-suppressive miRNAs targeting the MDM2/p53 axis were miR193a-5p and miR-146a-5p [59]. MiR-1249 is a direct transcriptional target of p53, and p53-mediated induction of miR-1249 inhibited tumor growth, metastasis, and angiogenesis in vitro and in vivo [60]. Ectopic expression of miR-133a markedly increased p53 levels and induced p21 transcription and, thus, significantly suppressed CRC cell growth in vitro and in vivo, and sensitized cells to doxorubicin and oxaliplatin [61]. The p53-induced miR-34 microRNA family mediated repression of c-Kit by p53 via a conserved seed-matching sequence in the cKIT 3 -UTR; ectopic c-Kit expression conferred resistance of CRC cells to 5-FU, whereas ectopic miR-34a sensitized the cells to the drug [62]. MiR-34a directly inhibited the oncogenic receptor tyrosine kinase CSF1R, and p53 repressed CSF1R by inducing miR-34a. Accordingly, resistance of CRC cells to 5-FU was mediated by miRNA-34a silencing (via CpG-methylation) and the resulting elevated expression of CSF1R [63]. In CRC cells, the transcription factor AP4 was downregulated by p53, which was indirectly mediated by the tumor-suppressive miR-15a and miR-16-1, targeting the 3 -UTR of AP4 mRNA, inducing mesenchymal-epithelial transition (MET) and inhibiting CRC cell migration and invasion [64]. MiR-16 repressed CRC cell growth by decreasing Survivin (BIRC5) expression through a direct targeting of BIRC5, and p53 negatively modulated BCL-2 by controlling miR-1915 [65,66]. Also, miR-148b, whose transcription is directly activated by p53, bound specifically to the 3 -UTR of P55PIK mRNA and suppressed p55PIK expression, which abolished proliferation and cell cycle progression of CRC cells and decreased tumor growth in vivo [67]. Furthermore, miR-143 and miR-145 function in a tumor-suppressive way, and the major mediators of the oncosuppression were genes belonging to the growth factor receptor-mitogen-activated protein kinase network and to the p53 signaling pathway [68]. In rectal tumors, elevated expression of miR-150-5p and miR-196b-5p significantly increased patients' survival [69]. In a genome-wide systematic approach, miR-30e, a direct transcriptional target of p53, was the most frequently deregulated miRNA in a p53-deficient background of CRC [70]. MiR-600 represents a direct negative regulator of p53 through binding the 3 UTR of the TP53 mRNA. Its overexpression decreased endogenous levels of mutant p53 and inhibited cell proliferation, migration, and invasion in mutant p53-expressing CRC cells [71]. Furthermore, the WNT/cMYC axis signaling inhibited the expression of p53 by promoting a direct targeting of p53 by miR-552 leading to resistance to drug-induced apoptosis, suggesting that miR-552 may function as an oncogene [72]. Furthermore, miR-27a was also identified to be oncogenic in CRC cells. The overexpression of miR-27a, i.e., its binding to two putative binding sites on the 3 -UTR of the TP53 mRNA, resulted in the decreased p53 expression [73]. MiR-300 was a direct positive regulator of p53 through binding to the 3 UTR of TP53 in mutant p53 CRC cells. Both miR-300 and p53 induced EMT, thus being oncogenic [74]. One more CRC-promoting miRNA described so far is miR-150-5p repressing the p53 pathway [75].
Micro-RNA 34a acts as translational regulator, which activates p53 by inhibiting its acetylation by MTA2 (Metastasis Associated 1 Family Member 2) and HDAC1 [55]. The methylation of miR34a correlated with the p53 wildtype status in CRC and other tumor types [56].

Methylation of the TP53 Gene
The TP53 gene is a direct target site for epigenetic regulation ( Table 3). The methylation inside and outside of exonic CG sequences of the TP53 gene was correlated with point mutations. Cigarette smoking increased the occurrence of methylation-associated mutations [79]. Methylation of the TP53 gene, histone modifications, chromatin remodeling, and non-coding RNAs were significantly associated with colitis-related carcinogenesis and tumor progression [80].
Polymerase epsilon (POLE) is a replicative polymerase important for efficient replisome assembly and strand synthesis, supporting tumor suppression [81]. POLE is involved in DNA repair and especially methylated cytosines (5mCs) are frequently mutated. Mutations in POLE exonuclease domain increase 5mC mutagenesis and a mutator phenotype. CRC with mutated POLE frequently contain highly methylated CpG islands in the TP53 gene [82].

Acetylation of p53
In addition to methylation, epigenetic regulation of p53 can also take place by acetylation (Table 3). NRDC is a histone-binding protein that binds HDAC1 and inhibits HDAC1 recruitment to the TP53 promoter and p53 acetylation [87,88].

Epigenetic Alterations Downstream of p53 4.1. Methylation Status of p53 Downstream Genes
Genes that are activated by p53 still can lead to inactive downstream signaling, if they are methylated (Table 4, Figure 2).

Methylation Status of p53 Downstream Genes
Genes that are activated by p53 still can lead to inactive downstream signaling, if they are methylated (Table 4, Figure 2). The BCL-2-interacting protein HRK interacts with BCL2 and is a pro apoptotic p53 target protein. Its expression can be inhibited by the methylation inhibitor 5-aza-2′-deoxycytidine, an effect which is further enhanced by histone deacetylase inhibitors (trichostatin A, depsipeptide). The methylation of the HRK promoter significantly correlated with the p53 wildtype status in 58 CRC cases [88].
The X-linked ectodermal dysplasia receptor (XEDAR) is a member of the tumor necrosis factor receptor family. P53 upregulates XEDAR expression through two p53-binding sites within intron 1 of the XEDAR gene. Inactivation of XEDAR results in enhanced cell adhesion and spreading, and resistance to p53-induced apoptosis. The expression of XEDAR was down-regulated by promoter hypermethylation or TP53 mutations in CRC cell lines and clinical biopsies [89].
The expression of the insulin-like growth factor-binding protein 7 (IGFBP7) is induced by binding of p53 to a p53-responsive element in intron 7 of the IGFBP7 gene. IGFBP7 methylation was significantly higher in 83 CRC biopsies than in normal colonic tissue. 5-Aza-2′-deoxycytidine restored p53-induced IGFBP7 expression. IGFBP7 methylation significantly correlated with wildtype p53 [90]. DLC1 (deleted in liver cancer 1) is a tumor suppressor. The DLC1-i4 is an isoform whose promoter is activated by wildtype p53. DLC1-i4 was methylated in 2/4 CRC cell lines (=50%), and demethylation by 5-aza-2′-deoxycytidine restored its expression [91]. The BCL-2-interacting protein HRK interacts with BCL-2 and is a pro-apoptotic p53 target protein. Its expression can be inhibited by the methylation inhibitor 5-aza-2 -deoxycytidine, an effect which is further enhanced by histone deacetylase inhibitors (trichostatin A, depsipeptide). The methylation of the HRK promoter significantly correlated with the p53 wildtype status in 58 CRC cases [89].
The X-linked ectodermal dysplasia receptor (XEDAR) is a member of the tumor necrosis factor receptor family. P53 upregulates XEDAR expression through two p53-binding sites within intron 1 of the XEDAR gene. Inactivation of XEDAR results in enhanced cell adhesion and spreading, and resistance to p53-induced apoptosis. The expression of XEDAR was down-regulated by promoter hypermethylation or TP53 mutations in CRC cell lines and clinical biopsies [90].

Regulation of p53 by acetylation
Several excellent reviews considered the various aspects of p53 acetylation. The acetylation of p53 at lysine 320 is achieved by histone acetyltransferase, PCAF (p300-CBP associated factor), which in most cases led to transactivation of the P21 (CIP1/WAF1/CDKN1A) promoter and increased histone acetylation after DNA damage [94,95]. Moreover, the addition of an acetyl group to p53 protein increased its stability, its binding to low affinity promoters, and other proteins [96]. On the other hand, inhibition of histone deacetylases (HDACs) resulted in increased p53 acetylation and eventually p53-dependent activation of apoptosis, cell cycle arrest, and senescence [97].

Regulation of Micro-RNAs by p53
Although not investigated to great detail, p53 can regulate micro-RNAs, which represents another level of gene regulation downstream of p53 (Table 4). P53 activated the transcription of miR-34 family members. The miR-34b/c CpG island is a bidirectional promoter, regulating the expression of both miR-34b/c and B-cell translocation gene 4 (BTG4). Methylation of this promoter silenced BTG4 transcription. The methylation of the miR34b/c CpG island was found in 100/111 CRC (=90%) [98,99].

Epigenetic Alterations and Clinical Outcome upon Standard Chemotherapy of CRC
In the past 10 years, a huge amount of data on epigenetic alterations in CRC has been published. Interest was placed in particular on putative clinical implications, emerging prognostic and diagnostic biomarkers, as well as on the implementation of the gained knowledge in precision (personalized) oncology [100][101][102][103][104][105].
Here, we focus on the impact of the CpG island methylator phenotype (CIMP), the most frequent epigenetically altered biomarker in CRC, on clinical outcome (disease progression, metastasis, and survival) of patients having received standard chemotherapy with 5-FU, oxaliplatin, and/or irinotecan. In a randomized controlled trial, significant differences in overall survival were not observed between patients with CIMP (+) vs. CIMP (−) tumors after adjuvant administration of oxaliplatin [106]. Thus, CIMP did not seem to be a prognostic biomarker in oxaliplatin-treated patients with resected CRC. On the other hand, in another clinical trial, CIMP was of prognostic value for stage III CRC patients treated with adjuvant oxaliplatin. A large cohort of well-defined patients with stage III CRC, CIMP (+) phenotype was associated with a shorter overall survival and a shorter survival after recurrence [107]. This was verified by another study, in which the CIMP (+) phenotype was an adverse prognostic marker in patients with metastatic disease treated with 5-FU/oxaliplatin and 5-FU/irinotecan [108]. Interestingly, in another clinical trial patients with stage III, CIMP-positive, MMR-intact CRC exhibited longer survival times, if irinotecan was added to combination therapy with 5-FU and leucovorin; i.e., those patients benefited most from the FOLFIRI protocol [109]. Furthermore, hypermethylation of p16 was predictive of clinical outcome in metastatic CRC patients treated with cetuximab and FOLFIRI, irrespective of KRAS mutation [110]. The progression-free survival of metastatic CRC patients with CIMP (+) tumors, who received sequential therapy with 5-FU/oxaliplatin (FOLFOX) as the first-line treatment, followed by irinotecan-based second-line treatment, was inferior to that of patients receiving the reverse sequence. It was concluded that the high mutation burden in EGFR-related genes in CIMP (+) tumors may cause a lower response to anti-EGFR antibody (cetuximab) therapy [111]. Recently, epigenetically regulated gene expression profiles revealed four molecular subtypes with prognostic and therapeutic implications in CRC [112]. A recent paper described a novel epigenetic signature of 8 hypermethylated genes that were able to identify metastatic CRC individuals with poor prognosis to oxaliplatin and irinotecan, characterized by CIMP (+) and MSI-like phenotype. The expression of the 8-genes signature and MSI-enriching genes was confirmed in oxaliplatin-and irinotecan-resistant CRC cell lines [113].

Conclusions and Perspective
While the significance of missense mutations in the TP53 gene has been recognized for a long time, the significance of epigenetic changes has only become clearer during the past few years. More and more data on epigenetic changes are being found not only in the TP53 gene itself, but also in genes upstream or downstream in the p53 signal transduction chain. Until now, it was assumed that about half of all CRC are affected by loss-of-function mutations in p53. If epigenetic changes upstream and downstream also have to be considered to contribute to CRC carcinogenesis, the significance of the entire p53 signaling pathway becomes much greater than previously assumed. This is not only of prognostic relevance for the survival time of CRC patients, but also plays a prominent role for the response to therapy, since p53 is not only a factor that promotes carcinogenesis and tumor progression, but also determines the success of chemotherapy and radiotherapy of tumors [114].
Many studies showed the importance of epigenetics of TP53 in cancer in general. The methylation of TP53 and p53-related genes were well studied in comparison to acetylation and micro-RNAs. Therefore, more research is required to better understand how the epigenetics of p53 contributes to CRC carcinogenesis.
The entirety of all partners involved in signaling is referred to as the "signalome". Therefore, we propose that the genetic and epigenetic characterization of the "p53 signalome" by the development of novel diagnostic devices based on biochips, DNA/RNAsequencing, and other advanced technologies have the potential to significantly improve today's diagnostic power in CRC management. For example, bisulfite treatment of genomic DNA in combination with high-throughput DNA sequencing enables the study of the genomic DNA methylation in general, and specifically, p53-dependent signaling pathway genes. This gold-standard (bisulfite-conversion) method has been adapted to fit with next-generation sequencing technologies and shotgun-sequencing approaches. Concerning the acetylation profile, the chromatin immunoprecipitation (ChIP) with high-throughput sequencing or DNA microarrays (ChIP-chip) is widely used.

Conflicts of Interest:
The authors declare no conflict of interest.