Open access peer-reviewed chapter

p53 Tumor Suppressor: Functional Regulation and Role in Gene Therapy

Written By

Zeenat Farooq, Shahnawaz Wani, Vijay Avin Balaji Ragunathrao, Rakesh Kochhar and Mumtaz Anwar

Submitted: 15 February 2022 Reviewed: 22 April 2022 Published: 01 August 2022

DOI: 10.5772/intechopen.105029

From the Edited Volume

p53 - A Guardian of the Genome and Beyond

Edited by Mumtaz Anwar, Zeenat Farooq, Mohammad Tauseef and Vijay Avin Balaji Ragunathrao

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p53, a homo-tetrameric protein found in mammalian cells, derives its name from the fact that it settles at around 53KDa position in SDS-PAGE, due to a “kink” in its structure. In its functional state, p53 forms a homo-tetramer and binds to the promoters of a wide array of genes. Binding of p53 downregulates the transcription of target genes. Most of the gene targets of p53 are involved in cell cycle progression, and therefore, any malfunctions associated with p53 have catastrophic consequences for the cell. The gene encoding for p53 known as TP53 is the most well-studied gene in the entire genome because of being the most highly mutated gene in all cancer types. It is due to this widely accepted and documented “cell protective feature” that p53 is generally referred to as “the guardian of the genome.” In this chapter, we will discuss the involvement of p53 in relation to carcinogenesis. We will also cover the major functions of p53 under normal conditions, major mutations of the TP53 gene, and their association with different forms of cancer.


  • TP53
  • DNA
  • caspases
  • cell cycle
  • apoptosis
  • mutations

1. Introduction

p53 is tumor-suppressor protein also named as “the guardian of the genome” since it prevents damaged DNA from getting inserted into genome and its proliferation in daughter cells. It is p53 that decides if DNA damage is to undergo repair or cell must undergo apoptosis or enter senescence when the damage is beyond repair. If possibility of repair exists, p53 activates other genes to fix the damage; otherwise, this protein prevents the cell from dividing and signals it to undergo apoptosis. By preventing division of cells with mutated or damaged DNA, p53 helps prevent the development of tumors. Named as p53 due to its migration at 53kd size in SDS-PAGE due to its structural confirmation, 53 kDa molecular mass, this protein was identified in 1973. Its gene TP53 is located on the short arm of chromosome 17 (17p13.1) in humans [1]. Total 393 amino acids constitute p53, which are distributed into three main domains, namely transcriptional activation domain, DNA-binding domain (DBD), and tetramerization domain. Transcriptional activation domain has role to stall RNA polymerase and activate the transcriptional machinery. DNA-binding domain binds to the specific regulatory sites on the DNA response elements and is more prone to mutations due to arginine/lysine residues due to abundance of lysine /arginine residues. The tetramerization domain functions as oligomerizer domain. The binding of tetrameric p53 via DBD to regulatory DNA motifs in the genome known as response elements with the consensus sequence RRRCWWGYYYN0–13RRRCWWGYYY (R = A or G, W = A or T, Y = C or T, N = any base) is the core event of the process. Various studies have reported dose-dependent target gene activation of p53, with high affinity of response elements for target p53 linked to cell cycle arrest and lower affinity linked to pro-apoptotic targets. This concept explains that cells undergoing feeble DNA damage are able to induce only low levels of p53, which can bind the high-affinity response elements, giving opportunity to cell for repairing its genome. However, if DNA damage is of higher level, higher p53 levels are generated to bind even the low-affinity response elements, which further activate pro-apoptotic target genes leading to cell death [2]. Mutations in p53 lead to cancers. Genome-wide analyses have shown that TP53 is the most frequently compromised gene in human cancer [3]. Efforts to reactivate p53 function in cancer have proven to be a successful therapeutic strategy in murine models and have gained attraction with the development of a range of small molecules targeting mutant p53. Either p53 can have loss of expression during cancers or express missense mutations causing a single amino acid substitution in otherwise full-length p53 proteins. Germline p53 mutations cause Li-Fraumeni syndrome (LFS) in patients, put them at risk of different cancers at a young age. DNA-binding domain (DBD) of p53 is most prone to somatic missense mutations [4]. Cancer-derived p53 missense mutants are impaired for most wild-type (WT) p53 functions. p53 also acts as a transcription factor to control the expression of several coding and noncoding RNAs and genes including p21, MDM2, GADD45, PERP, NOXA, and CYCLIN G. In addition, p53 also suppresses the expression of some genes, such as MAP4 and NANOG [5]. Also, it interacts with cytoplasmic and mitochondrial proteins to directly modulate their activity [6]. The posttranslational modifications of p53 play important roles in dictating the cellular responses to various stresses. For example, the phosphorylation of p53 at Ser46 primarily activates p53-dependent apoptosis after DNA damage. In addition, the phosphorylation of p53 at Ser315 is important for suppressing NANOG expression during the differentiation of ESCs. The p53 activity can also be modulated by protein–protein interaction. For example, the ASPP family proteins promote the p53-mediated apoptosis by enhancing p53-dependent induction of pro-apoptotic genes such as PUMA [7]. The importance of the transcriptional activity of p53 in tumor suppression is further underscored by the findings that the hotspot missense mutations of p53 in human cancers uniformly disrupt the normal DNA-binding activities of WT p53. In addition to the loss of WT p53 activity, p53 mutants also gain oncogenic activities in promoting tumorigenesis [8]. p53 has major role in detection of stress pathways, such as hypoxia and metabolic stress. In response to genotoxic and oncogenic stresses, p53 induces cell cycle arrest, apoptosis, or senescence of the stressed somatic cells to prevent the passage of the genetic abnormalities to their off springs, thus maintaining the genomic stability of mammalian cells [9]. In addition, p53 plays complex roles in cellular metabolism, contributing to p53-dependent genomic stability and tumor suppression [10]. In addition, the protein levels of p53 are also maintained at low concentration in the absence of stresses, because several E3 ligases such as MDM2 form complex with p53, leading to the ubiquitination and degradation of p53. Therefore, as potent negative regulators of p53 stability and activity, MDM2 and MDMX are oncogenes often overexpressed in human cancers to inhibit p53 function [11]. In addition to its role in cellular stability, role of p53 in embryonic stem cells (ESCs) has also been elucidated. Expansion of ESCs for dozens of passages prior to their differentiation into lineage-specific functional cells is required to harness their potential to be used in clinics for addressing different issues. Clinical potential. Thus, its highly prevention of DNA damage and activation of oncogenic pathways are much prone to self-renewal and differentiation of ESCs. Role of p53 comes to play for maintaining the genomic stability of hESCs. However, in contrast to somatic cells, ESCs lack p53-dependent cell cycle G1/S checkpoint, apoptosis, and senescence. Instead, when activated, p53 induces the differentiation of ESCs by directly suppressing the expression of the critical pluripotency factor Nanog. Thus, ESCs with unrepaired DNA damage or oncogenic stress are eliminated from the self-renewing pool due to the reduced Nanog expression, hence ensuring the genomic stability of self-renewing ESCs [12]. p53 is thus thought to induce the expression of the differentiation-related genes and downregulate the pluripotency genes in response to DNA damage in ESCs. In the absence of stresses, the activity of p53 must be suppressed to maintain pluripotency. The key pluripotency factor OCT4 activates the expression of histone deacetylase SIRT1, which inactivates p53 by deacetylation of p53 (13). The extensive culture of hESCs might accumulate hESCs harboring mutated p53, raising cancer risk upon long-term culture [13]. The p53 mutants might lead to gain of functions to promote the expression of pluripotent genes and thus the preferential expansion of hESCs harboring these p53 mutants [14]. Therefore, culture conditions that can avoid the favorable selection of hESCs harboring p53 mutations during the extended culture are required to maintain healthy ESCs. p53 also has role in inducible pluripotent stem cells (iPSCs). Biggest limitation of iPSC technology is the extremely low efficiency of successful reprogramming. p53 has been discovered to have corner stone role for reprogramming [15]. Reprogramming factors are especially c-Myc and Klf4 that are potent oncoproteins, which are often overexpressed in human cancers. The overexpression of such oncoproteins in somatic cells will activate p53, which can all block successful iPSC reprogramming and suppress the expression of Nanog that is required for maintaining pluripotency. Therefore, the silencing of the p53 gene during reprogramming has become an effective approach to increase the reprogramming efficiency [16]. In addition, proteins such as Oct4 and ZSCAN4 can promote the reprogramming efficiency by inhibiting p53 [17]. The silencing of the genes that are responsible for p53-dependent cell cycle arrest and apoptosis, such as p21 and Puma, can also increase the frequency of nuclear reprogramming into induced pluripotency. On the other hand, the critical roles of p53 in maintaining genomic stability of mammalian cells raise a serious concern for the genomic instability of iPSCs as iPSCs harbor increased genetic abnormalities. First iPSC-based clinic trial to treat macular degeneration was put to halt due to high accumulation of genomic instability [13]. The genomic instability can also contribute to the immunogenicity of iPSC-derived autologous cells [18, 19]. The optimization of the reprogramming technology and the culture conditions of PSCs is required to improve PSC-based human cell therapy.


2. Stability of p53

p53 functions as a “genomic guardian” that regulates downstream targets responsible for cell fate control. p53 prevents various types of stresses such as DNA damage, hypoxia, metabolic stress, from expressing their consequences on genome, and progeny of new cells [20, 21]. The activity of p53 is tightly regulated by a complex network that includes an abundance of stress signals, posttranslational modifications, and various signaling pathways. Under normal cellular physiology, p53 is a short-lived protein and expressed at very low levels. Under stressful conditions, p53 is accumulated in the cell, and its degradation is prevented [22]. The stability of p53 is controlled predominantly by several E3 ligases, including the major ligase MDM2-mediated ubiquitination and subsequent proteasome-dependent degradation by the 26S proteasome [23, 24]. p53 levels remain low in normal non-stimulated cells. MDM2 maintains level of p53, by promoting the polyubiquitination of p53 and its degradation by proteasome pathway [25]. The major ubiquitination sites of p53 mediated by MDM2 are six lysine residues at the carboxy terminus (K370, K372, K373, K381, K382, and K386) [26]. Further, p53 is negative regulator of the MDM2. Increased p53 levels can induce MDM2 expression, leading to a decrease in p53 expression [25, 26]. MDM4 is similar to MDM2 and inhibits p53-mediated transactivation. Inhibition of MDM2 and MDM4 causes accumulation of p53 and its activity. p53 expression is also induced upon its release from its negative regulatory factors. Ubiquitination is another mechanism to prevent p53 from binding to the downstream targets, leading to apoptosis and cell cycle arrest [27, 28]. Ubiquitinylation agents of p53 have been identified, such as Pirh2, ICP0, COP1, TOPORS, ARF-BP1, CHIP, Ubc13, synoviolin, EF41, CARP2, WWP1, MSL2, E6-AP, TRIM2454, and MKRN1 [29]. Ubc13, WWP1, E4F1, and MSL2 are E3 ligases. Besides these E3 ligases, MDM2 at low level also mediates mono-ubiquitination of p53, causing proteasome-independent p53 ubiquitination [30]. Type of ubiquitination on p53 determines its effects on p53 function. E3 ligases and MDM2 can mediate lysine-48-linked polyubiquitination of p53 and target it to the 26S proteasome for degradation. Other types of ubiquitination, including mono- or lysine 63-linked polyubiquitinations, regulate nuclear export and cytosolic localizations of p53 [31].

Upon DNA damage, the interaction between p53 and MDM2 is suppressed, resulting in increasing levels of p53 protein and transcriptional activation of p53 target genes [32]. Stress signals activate ATM kinase and the DNA-PK. These kinases are starters of signal transduction cascades, which phosphorylate the N-terminus of p53 and the C-terminus of MDM2. They further dephosphorylate the central domain of MDM2, leading to weakening the interaction of p53 and MDM2 [33, 34, 35]. This prevents degradation of p53 and its accumulation to act on stress-induced damage. Oncogenes such as Myc or Ras also act as signals to stabilize p53, but they use a different route. They induce expression of p14/16ARF, which binds to MDM2, inhibits its ubiquitin ligase activity, sequesters MDM2 in the nucleolus, and promotes MDM2 degradation [36, 37].

However, proteasomal degradation of p53 also has been shown to occur independently of MDM2 if newly synthesized p53 is being intrinsically unstructured [38, 39, 40]. The mechanism behind the MDM2-independent system involves Isg15-modifying system. The system is associated with the translational machinery and targeting of newly synthesized proteins [41, 42]. Different types of stimuli induce Isg15, and these include type 1 IFNs, lipopolysaccharide, and viruses [43]. Identified as a p53 target, Isg15 is also induced during the chemotherapy and requires functional p53. ISGylation is a process similar to ubiquitinylation wherein conjugation to the target proteins occurs in a three-step cascade mechanism. UBE1L is Isg15-activating E1, UBCH8 is E2 Isg15-conjugating enzyme and Isg15 E3 ligase with HERC5 being the main E3 ligase for Isg15. ISGylation negatively regulates the ubiquitin-proteasome pathway by direct interference with polyubiqutination, providing evidence of potential cross talk between these two systems [44]. It has been found that p53 is efficiently ISGylated by HERC5 and subsequently degraded by the 20S proteasome. Furthermore, Isg15 deletion increases the misfolded, dominant-negative p53, so it has been proposed that ISGylation is likely to work as a signal for degradation of misfolded p53, and this regulation is important for p53-mediated biological function [45].

The stability of both wild-type p53 and mutant p53 has been shown to be regulated by lipid messenger phosphatidylinositol 4,5-bisphosphate (PI4,5P2) PI4,5P2 directly binds to protein targets known as PI4,5P2 effectors and regulates their function by modulating activity and localization. The majority of PI4,5P2 is generated by phosphorylation of PI4P and PI5P by type I and type II phosphatidylinositol phosphate kinases (PIPKs), respectively, and each type has α, β, and γ isoforms in humans. PI4,5P2 is also found in the nucleus. Nuclear PI4,5P2 is distinct from the nuclear envelope and is found in non-membranous structures such as nuclear speckles [46, 47, 48]. p53 associates with PI4,5P2-generating enzyme, type Iα phosphatidylinositol-4-phosphate 5-kinase (PIPKIα) in the nucleus, and of PIPKIα diminishes p53 stability. Moreover, PI4,5P2 generated by PIPKIα interacts to p53 to promote binding of HSP27 and αB-Crystallin. Both PI4,5P2 binding and recruitment of HSP27 are required for stabilization of nuclear p53. Thus, PIPKIα and the PIPKIα-p53-PI4,5P2-sHSP complex have been reported as promising therapeutic targets in cancer [49].

Notably, other posttranslational modifications, such as acetylation, methylation, neddylation, and sumoylation, play important roles in regulating p53 transcriptional activities. p53 is among the first non-histone proteins known to be regulated by acetylation and deacetylation [50, 51]. There are more than 50 sites in p53, which are regulated through posttranslational modifications such as phosphorylation, acetylation, methylation, and so on. These modifications have been shown to play role in regulating the stability of p53. Phosphorylation of p53 at serine and threonine residues of its N and C terminal regions takes place as a result of cell stimulation. Some of the phosphorylation sites, however, are phosphorylated in unstimulated cells and become dephosphorylated as a result of DNA damage [52]. The quintessential phosphorylation in p53 takes place at serine 15 residue and induces its dissociation from MDM2, resulting in its stability and activation of downstream functions, whereas phosphorylation at position 392, induced by DNA damage, plays a role in activation of sequence-specific DNA-binding property of p53. Phosphorylation also plays a role in formation of functionally active tetramers of p53. The transactivation domain (TAD) of p53 forms two domains TAD1 and TAD2. TAD2 interacts with the p62 family of transcription factors, which initiate chromatin decondensation at promoters. Similar to phosphorylation, acetylation of lysine residues of p53 plays role in a variety of functions through its stabilization and activation. Acetylation of p53 inhibits cell cycle progression at G2 phase and SIRT1deacetylase interacts with p53 in the nucleus, specifically deacetylating the K382 acetylation of p53 [53]. Different stimuli induce p300-mediated acetylation of lysine residue 305, both in vitro and in vivo. Lysine K320 acetylation plays role in regulation of p53 shuttle between nucleus and cytoplasm. It is also involved in BAX-mediated apoptosis after DNA damage in intestinal adenomas. In addition to acetylation, methylation of lysine and arginine also regulates p53 function. For example, K372 methylation enhances p53 stability, increases its binding to chromatin, and promotes transcriptional activity, whereas K370 methylation inhibits transactivation of p53. Additionally, arginine methylation also acts as an important regulatory mechanism for modulating p53 activity. As a result of DNA damage, p300 recruits arginine methyltransferase PRMT5 to p53, which helps in the oligomerization of p53 to modulate its transcriptional activity, whereas lack of PRMT5 alters specificity of p53 binding and triggers apoptosis. Also, siRNA-mediated knockdown of PRMT5 reduces the protein levels of p21, one of the downstream targets of p53. Mutations in the arginine residue Arg 337 are also known to be related to development of tumor and changes in biochemical characteristics of p53 oligomers. Further studies into the role of arginine mutations and enzymes, which methylate these residues (arginine methyltransferases), can lead to exploration of novel mechanisms of p53 regulation and function [54].


3. Role of p53 in carcinogenesis

p53 is involved in mitigating cellular stress such as hypoxia, DNA damage, and oncogene activation by initiating stress response mechanisms that play role in preserving genome integrity. p53 protein is famously known as the “tumor suppressor p53” because the normal functioning of p53 acts as a huge roadblock to cancer initiation and progression [55, 56]. Therefore, for carcinogenesis to take place, mutations in the p53 gene TP53 are required, which can have a significant impact on the function of p53. This is part of the reason that p53 is one of the most frequently mutated proteins in all cancers, with the numbers being as high as 53% frequency of p53 mutations in all cancers. We will discuss some of the most important p53 mutations and their effects on carcinogenesis in the next section.

3.1 Loss of p53 function

p53 is known to act as a transcription factor by binding to various DNA response elements in the target sequences. More than 100 response genes of p53 have been identified, which include CDKN1A (p21 encoding gene), BBC3, PERP, and BAX (apoptosis genes), THBS1 (angiogenesis gene), and so on [57]. Of all the domains of p53 protein, DNA-binding domain is very critical in mediating its interaction with response elements. Therefore, base mutations in sequence of DBD (mis-sense mutations) are linked with tumorigenesis as they lose the ability of interacting with DNA elements involved in tumor progression such as proto-oncogenes. These mutations arise in somatic cells either spontaneously or secondary to DNA damage. However, not all mutations affect the function of p53 in a similar manner, and the extent to which a mutation can affect tumor progression depends upon the residue being mutated and the region of gene carrying this mutation. The sequence of response elements within the target genes also determines level of p53 binding. Some of the tumor types carry mutations, which lead to a gain of function of p53 agonists such as Mdm2 and Mdm4. This results in suppression of p53 activity irrespective of the availability of normal levels of wild-type p53 in the cell. Mdm2 is a ubiquitin ligase, which binds to p53 and targets it for proteasomal degradation under normal conditions to maintain p53 at a low level in normal cells. MDM2 also binds to p53 mRNA to regulate its translation [58]. On the other hand, MDM2 itself is induced by p53, and therefore, the two proteins regulate each other through a negative feedback system. Upon receiving a stress stimulus such as DNA damage, p53 is post translationally modified by a variety of upstream effectors. These modifications inhibit the association of p53 and MDM2 to allow p53 binding to DNA response elements to initiate a stress response pathway. P53 response genes include cell cycle control genes, apoptotic genes, cellular senescence genes, and others. p53 mutations also arise in germline cells in individuals with Li Fraumeni syndrome and lead to an increased risk of developing adrenocortical, brain, and breast tumors [59].

3.2 Mutations in p53

Most of the mutations in TP53 are intronic, with no established role in tumorigenesis. Only 19 of these mutations are exonic among which 11 are nonsynonymous (replacement of one amino acid with another as a result of base change) and four are synonymous (replacement of a codon with another coding for the same amino acid). Molecular evidence suggests that P47S [60] and R72P [61] mutations lead to changes in p53 binding to response elements. Polymorphisms also exist in the response element sequences of p53 target gene promoters, which can alter the binding of p53 up to 1000-fold [62]. p53 mutations have also been identified in 50% of adult neoplasia including the colon, lung, esophagus, stomach, liver, breast, and uterine cervix; however, no molecular data are available so far to explain the mechanism behind these mutations [63]. Also, p53 mutations occur more frequently in carcinomas than adenomas, suggesting that these represent a late event in clinical carcinogenesis. Some synonymous mutations have also been shown to alter binding of p53 mRNA to MDM2.

3.3 p53 protein: Protein interactions and carcinogenesis

Apart from DNA binding and transcriptional control, p53 also binds directly to various proteins to exhibit its tumor suppressor activity. These include cell cycle control, DNA repair, and apoptotic genes [64]. It binds to Bcl-2 family of proteins (pro-apoptotic proteins) in cells with damaged DNA to release intermembrane molecules of mitochondria and triggers apoptosis [65]. Tumor-associated mutations in p53 also affect its protein–protein interactions to promote carcinogenesis.

3.4 Role of posttranslational modifications of p53 in carcinogenesis

p53 can be modified by a variety of posttranslational modifications such as phosphorylation, acetylation, methylation, and ubiquitination on multiple residues [66, 67]. These modifications could alter the ability of p53 binding to response elements, protein–protein interactions as well as stability [68]. The first step toward p53 activation is phosphorylation of p53 at residues S10, S20, and T18 by a range of upstream kinases such as ATM and DNA-PK to increase its stability by abrogating its interaction with Mdm2. The choice of the residue targeted for phosphorylation depends on the upstream kinase and the pathway being activated. Activated p53 acts as a transcription factor by binding as a tetramer and phosphorylation at S392 at its C-terminal enhances the stability of p53 tetramer. Epigenetic modifications are also known to regulate p53 activity with both activating and repressive effects. Increase in methylation of p53 promoter decreases its rate of transcription [69] while acetylation of p53 by CBP/p300 increases its activity by inhibiting its binding with MDM2 [70].


4. p53 as tumor suppressor and DNA damage sensor

In the year 1989, the independent studies led by Bert Vogelstein and Joh Minna for the first time reported the presence of p53 mutations in colorectal and lung cancer cells [71, 72]. These studies emphasized that the genetic abnormalities of p53 show gross changes such as homozygous deletion and abnormally sized messenger RNAs along with a variety of point or small mutations and change of amino acid sequence in the region highly conserved between mouse and human. These studies were subsequently confirmed by other groups stating the importance of p53 gene in various cancer types [73, 74, 75, 76, 77]. However, the tumor suppressor function of p53 was first confirmed by Stephen Friend group in 1990, in which, they demonstrated the existence of somatic and germline p53 mutations in families with Li-Fraumeni syndrome, in which affected members are genetically predisposed to cancer. In all the families studied, there was a close correlation between transmission of the mutant allele and development of cancer. There are currently more than 55,000 literature reports in various types of human cancer [78, 79, 80].

The p53 tumor functions are influenced by several factors such as cell type, tissue microenvironment, and oncogenic events acquired during the tumor initiation. The activation of p53 can occur in response to DNA damage, oncogene activation, and hypoxia, in which p53 subsequently orchestrates the biological outputs such as cell-cycle arrest, senescence, apoptosis, and autophagy modulation (Figure 1).

Figure 1.

Role of p53 in tumor suppressor function. The DNA damage signal sensed by ATM/ATR and oncogene activation leading to MDM2 inhibition resulting in the activation of secondary sensor p53 to orchestrates, cell cycle arrest/DNA repair/senescence/apoptosis.

4.1 The tumor suppressor function of p53 in response to cellular stress comprises three basic steps

4.1.1 Stabilization of p53

Mouse double minute2 homolog (MDM2) plays an important role in negatively regulating p53 function. Hence, the initial stabilization phase of p53 attained through actions that disrupt its interaction with MDM2. Posttranslational modification of p53 such as the amino-terminal phosphorylation by various cytoplasmic kinases prevents the binding of MDM2, which results in stabilization of p53 in response to DNA damage from ionizing radiation or certain chemotherapeutic agents (Figure 1) [81, 82].

4.1.2 Sequence-specific DNA binding

Once p53 is stabilized, it will bind to DNA in a sequence-specific manner. The p53 protein consists of a carboxy-terminal basic DNA-binding domain, and majority of the tumor-associated mutations in p53 protein occur in this domain, hence it is the “hot spot” for mutations. The ubiquitous DNA-binding activity of carboxy-terminal domain of p53 is to assist DNA binding and the search of p53 target sites subsequent to cellular stress [83, 84, 85].

4.1.3 Transcriptional activation of target genes

After stabilization of p53 and sequence-specific DNA binding, p53 activates to repress its target genes. p53 promotes transcriptional activation or repression of target genes by interacting with general transcription factors such as Transcription factor II D (TFIID) or TBP-associated factors (TAFs) depending on the complexity of promotor selection. Recent studies have reported that posttranslational modifications of p53 can influence the recruitment of p53-binding proteins to specific promoters. The interaction of CBP/p300 with p53 results in the posttranslational modifications such as p53 acetylation along with histone acetylation leading to more open chromatin conformation near p53 targets and more active p53 protein [86, 87, 88].

4.2 p53 as a DNA damage sensor

Internal cellular responses and signal transduction to genotoxic stress resulting in the activation of various transcription factors are a very complex process starting with “sensing” of the DNA damage [88]. There have been extensive studies across various disciplines exploring the activity of p53 in sensing the DNA damage induced during genotoxic stress [89]. Identifying the specific residues modified on p53 in response to DNA damage has allowed a greater understanding of the molecules that may signal to p53. Initial studies showed that the DNA damage response was sensed by p53 via Phosphoinositide 3 kinase (PI-3k) and PI 3 kinase like family members as being instrumental in mediating phosphorylation of serine-15 and regulating p53 in response to DNA damage [90, 91, 92]. Despite mediating intracellular singling events by way of phosphorylating inositol lipids, PI-3k family members have recently been expanded by the identification of their role in phosphorylating p53 to sense DNA damage. Members of this subfamily include the catalytic subunit of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ataxia-telangiectasia mutated (ATM), ATM Rad3-related (ATR), and Transformation/Transcription domain-associated protein (TRRAP) [93, 94, 95, 96, 97, 98, 99, 100]. Overall, p53 also functions as sensor of DNA damage by working with aforementioned kinase family members.


5. Role of p53 in apoptosis and cellular stress

During homeostatic condition, the nontransformed cells express very low or often undetectable amount of p53 protein, whereas it may still show readily detectable mRNA expression. In naïve cells, the level of p53 protein is very unstable with a half-life ranging from 5 to 30 min, owing to continuous degradation largely mediated by MDM2. The MDM2 was identified as oncogene formed as a complex with p53 protein for the first time in 1992 [101]. Ever since, persuasive evidence has emerged for MDM2 to have a physiologically critical role in controlling p53. Many findings emphasized that MDM2 itself is the product of a p53-inducible gene [25102, 103]. Thus, both MDM2 and p53 are linked to each other through an autoregulatory negative feedback loop intended at maintaining low cellular p53 levels under naïve condition. MDM2 is known to harbor a p53-specific E3 ubiquitin ligase activity within its evolutionarily conserved Zinc-binding domain, which is critical for its E3 ligase activity [104]. Studies have shown that MDM2 is largely expressed in nucleus and bound to p300/CBP leading to the p53 ubiquitination [105]. The crystallographic studies showed the biochemical basis of MDM2-mediated inhibition of p53 function, in which, the amino terminal domain of MDM2 forms a deep hydrophobic cleft into which transactivation domain p53 binds, thereby concealing itself from interaction with the transcriptional machinery [106].

During cell transformation, caused due to stress stimuli such as activation of oncogenes or any other DNA damage signals, the p53 protein level substantially increases because of the activation of survival pathways, which lead to the inhibition of MDM2 and posttranslational modifications in the p53 protein itself. The activated p53 thus turns on the diverse cellular effector process, including cell cycle arrest, cellular senescence, DNA damage-repair pathways, and apoptotic cell death [25, 107, 108, 109, 110, 111].

In 1991, studies from Moshe Oren group showed for the first time that p53 can induce apoptosis of myeloid leukemia cells. They showed that temperature-sensitive conditionally active mutant of p53, in which at 37°C behaves as mutant but at 32°C it assumes wild-type (WT) p53 structure and function and starts inducing apoptosis of leukemia cells in vitro [112]. Further these studies using temperature-sensitive p53 or WT p53 were confirmed by various other groups in different cancer cell lines such as erythroleukemia, colon cancer, and Burkitt lymphoma [113, 114].

5.1 P53-mediated intrinsic apoptotic pathways

Broadly, the mammalian cells endure apoptosis in two distinct manners. Intrinsic or mitochondrial stress apoptotic pathway, activated during stress conditions, such as cytokine deprivation, ER stress, or DNA damage, which is regulated by B-Cell Lymphoma 2(BCL-2) family proteins. Another mechanism of apoptotic activation of mammalian cells due to ligation of members of tumor necrosis factor receptor (TNFR) family bearing intracellular death domain, called as extrinsic apoptotic pathway [115].

The intrinsic apoptotic pathway or mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, apoptosis-inducing factor (AIF), Smac (second mitochondria-derived activator of caspase)/DIABLO (direct inhibitor of apoptosis protein (IAP)-binding protein with low PI), Omi/HtrA2 or endonuclease G from the mitochondrial intermembrane space. The release of cytochrome c into the cytosol triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex, whereas Smac/DIABLO and Omi/HtrA2 promote caspase activation through neutralizing the inhibitory effects to the IAPs (Figure 2) [116, 117].

Figure 2.

P53-mediated apoptotic pathways and their cross talk. Extrinsic apoptotic pathways mediated by p53 via activating caspase 8 leading to the activation of Caspase3 and 7. Intrinsic apoptotic pathway mediated by p53 leading to the activation of pro-apoptotic molecules BH3 only proteins and BAX/BAK leading to the activation of caspase 9 via mitochondrial mediated cytochrome c, further activating caspase 3 and 7 to induce apoptosis.

In vitro studies using overexpression of WT p53 or temperature-sensitive p53 showed that elevated expression of anti-apoptotic BCL-2 could prevent p53 induced apoptosis. Further, the cells rescued from p53-induced apoptosis by elevated expression of BCL-2 still able to perform cell cycle arrest, indicating BCL-2 does not directly block p53, hence p53 was fully functional. Thus, BCL-2 and its family member proteins inhibit the p53-induced apoptosis at a downstream point of apoptosis signaling, suggesting induction of cell cycle arrest and apoptosis by p53 through distinct pathways [118, 119]. Though, the study using overexpressed WT and temperature-sensitive p53 mutants provides efficient evidence for apoptosis induction, it does not resemble the physiological condition where p53 protein level is normal [111]. All these caveats are addressed once after the generation of p53 knockout animals, in which, the thymocytes and other lymphoid cell subsets are completely resistant to apoptosis induced by γ-radiation and treatment with chemotherapeutic drugs that induce DNA damage.

The most intuitive link between p53 and BCL-2, unveiled the quest to identify the p53 activated initiators of the cell death pathways that is regulated by BCL-2. Many downstream effectors were identified in response to overexpression of p53 at both physiological and nonphysiological level. These include BCL-2 associated X protein (Bax), BCL-2 homolog 3 (BH3)—only proteins, p53 upregulated modulator of apoptosis (PUMA), and BH3 interacting domain death agonist (Bid). Gene targeting studies in both in vivo and in vitro showed that pro-apoptotic members of BCL-2 family can act downstream of p53 during apoptosis. The studies using Bax-knockout mouse embryo fibroblast showed that they are desensitized to oncogene-induced and p53-dependent apoptosis leading to suppression of tumorigenesis [120]. Some in vitro studies showed that knocking down either Bax or PUMA in cell lines induces various levels of apoptotic defects [121, 122, 123, 124, 125]. Taken together, these studies suggest that loss or mutation of p53 attenuates the expression of the downstream targets implying that the phenotypes of the attenuated effectors show defects in p53-mediated apoptosis.

Though, p53-mediated intrinsic pathway of apoptosis controls the factors that act upstream of the mitochondria, it can also transactivate several components of the apoptotic effector machinery [124]. Apoptosis protease activating factor 1 (Apaf-1), caspase-6, and E2 factor family transcription factor (E2F) are the apoptotic effectors regulated by p53. Apaf-1 is known to act as coactivator of caspase-9 and helps initiate caspase cascade, and p53 loss can interfere in Apaf-1-mediated caspase cascade initiation [126]. In addition, p53 can upregulate the caspase 6, which is known as an effector caspase, leading to enhanced chemosensitivity of some cell types [127]. Likewise, p53 interferes with E2F and could result in promoting apoptosis and increase the caspase expression through a direct transcriptional mechanism [126].

5.2 p53-mediated extrinsic apoptotic pathways

Extrinsic apoptotic pathway also known as death receptor-mediated apoptotic pathway occurs under stress condition due to stimulation of death receptors of the tumor necrosis factor (TNF) receptor superfamily such as CD95 (APO-1/Fas) or TNF-related apoptosis-inducing ligand (TRAIL) receptors result in activation of the initiator caspase-8, which can propagate the apoptosis signal by direct cleavage of downstream effector caspases such as caspase-3 (Figure 2).

Several studies support the hypothesis of p53-mediated intrinsic pathway of apoptosis, few also showed that the extrinsic apoptotic pathway can also regulated by p53, although the overall contribution of p53-mediated extrinsic apoptotic pathway remains debatable and is still being researched [124]. Some of the proteins that are involved in extrinsic mechanisms such as Fas/CD95, Fas ligand, and death receptor 5 (DR5) are shown to be direct targets of p53 [128, 129, 130]. Moreover, some studies have also shown that there is a cross talk between intrinsic and extrinsic pathways because of the ability of p53 to transactivate Bid [125]. Consequently, p53 may sensitize cells to death receptor ligands, either inducing apoptosis directly or enhancing cell death in ligand-rich environment. Some studies have shown that disabling p53 sensitization to death receptor ligands by mutating p53 can promote drug resistance and can provide an ambient tumor microenvironment with immune privilege.

Despite being directly involved in both intrinsic and extrinsic apoptotic pathways, p53 can also regulate the survival signals indirectly. Phosphatase tensin homolog at chromosome 10 (PTEN) is a lipid phosphatase, known to inhibit phosphoinositide 3-kinase induced survival signaling by dephosphorylating 3′-phosphorylated phosphatidylinositides (PIP3) to 2′-phosphorylated phosphatidylinositides (PIP2). Some studies suggest that p53 can interfere in survival signaling by transactivating the PTEN promotor leading to increased expression of PTEN. Although, the disruption of PTEN can compromise p53-mediated apoptosis [131, 132]. Independent studies from Puzio-Kuter et al., and Freeman et al., showed that the tumor suppressor PTEN regulates the activity of p53 and levels of p53 levels through mechanisms involving both phosphatase dependent and independent manner [131, 132]. Thus, in the case of PTEN mutation or loss, p53 can neutralize the survival signals, seemingly diminishing the threshold needed for proapoptotic factors to trigger cell death.


6. Role of p53 in gene therapy

Most of the human cancer types show altered p53 level, and hence, the concept of restoration of p53 for cancer therapy is a very attractive strategy. The normal function of mutated p53 can be restored using various pharmacologically active small molecular inhibitors, by inducing massive apoptosis or reactivating p53. The compounds such as PRIMA-1, CP-31398, and SH group targeting compounds induce the apoptosis and reactivate the p53 [133, 134]. Another set of small molecular inhibitors such as Nutlin-3a, RG7112, CGM097, and SAR405838 block the interaction of p53 with MDM2 [135, 136, 137]. Though, the abovementioned small molecular inhibitors have shown good anticancer efficacy, they also have some limitations. For instance, it is still unclear whether PRIMA-1 and similar compounds effectively target all mutant p53 variants and whether the tumor suppressor functions of p63 and p73 could be negatively affected [133]. Similarly, the compounds inhibiting p53 and MDM2 interactions are not shown to be effective in tumors with a high prevalence of p53 mutations. All these impediments with p53 target using pharmaceutical agents paved a new way toward gene therapy. Scientists around the globe developed various techniques in the field of biotechnology to deliver the healthy, normal functioning p53 gene to cells that turned into cancerous due to the mutations in the p53 gene. Different mode of p53 gene therapy using different vectors is discussed below.

6.1 Nonreplicating viruses-based p53 gene therapy

This process involves a healthy p53 genes and a vehicle viral vector, in which the viral DNA has been altered to prevent it from replicating. This “safe version” of viral vector is then used to transport healthy p53 into transformed cells by directly injecting into the tumor site. If the transduction is successful, the p53 gene will make a functional p53 protein within the tumor microenvironment apparently restoring the normal p53 cellular function and thereby preventing cancer growth [138]. The history of p53 gene therapy is quite interesting. In 1992, Dr. Jack A Roth from MD Anderson Cancer center led a team to investigate the first p53 gene therapy clinical trials, which were approved by the National Institute of Health (NIH) and US Food and Drug Administration (FDA). He demonstrated and proved the gene therapy efficacy of p53 through laboratory and preclinical studies, which led to the approval for this historic protocol. In brief, they developed a retroviral and adenoviral vector expressing p53 tumor suppressor gene and completed the first clinical trials in lung cancer patients against non-small-cell lung carcinoma, by showing that restoration of function for a single tumor suppressor gene could mediate regression of human cancer in vivo [138, 139, 140, 141]. Adenovirus p53 became the first gene therapy approved for human use. Ever since, thousands of patients received different p53 mediated gene therapy with replication deficient viral vectors under many clinical trials without any significant adverse effect.

6.2 Oncolytic virus-based p53 gene therapy

The oncolytic virus therapy utilizes replication-competent viruses to kill malignant cells, leaving normal cells unscathed. Studies have shown limited success using a modified form of the measles virus to target the mesothelioma cells using oncolytic viruses alone [142]. Researchers hope that combining p53 gene with oncolytic viruses to make the treatment more effective than gene therapy or oncolytic therapy alone and these studies are still under preclinical trial stage. The preclinical studies combining p53 gene therapy along with oncolytic viruses suggested that the expression of WT p53 transgenes improves the oncolytic virus therapy safety, onco-selectivity, increases onco-toxicity, and augments antitumor effects by promoting the stimulation of anticancer immune responses [143].

6.3 Nanoparticles-based p53 gene therapy

Another easy and convenient method of delivering p53 gene is using synthetic nanoparticles. Like viruses, nanoparticles could be designed to deliver their contents to cancer cells specifically, leaving healthy cells unaffected. Nanoparticle-based p53 gene therapy is still under clinical trials, and these studies suggested that the nanoparticle tagged with p53 gene would be a safer mode of gene delivery method compared with viruses, creating no risk of infection, and they could travel through the body without provoking a response from the immune system [144, 145, 146].


7. Conclusion

Most of the “tumor associated mutations” in p53 are single base substitutions in the coding sequence [147]. Apart from these, more than 200 single nucleotide polymorphisms have been identified in TP53 with no measurable consequence on p53 function and/or tumor progression. p53 is unique for being the most well-studied and most frequently mutated tumor suppressor gene with a wide spectrum of residual activity as a direct consequence of the mutated residue [148]. The molecular basis behind most of the p53 mutations is not well understood. Therefore, better understanding of these mechanisms could lead to improvements in clinical treatment of cancers carrying p53 mutations. Recently, other mechanisms such as micro RNAs have been implicated to be involved in p53-mediated gene regulation and which further necessitates undertaking more studies to understand the roles of the “guardian of the genome” in a more elaborate manner. Because of the diversity of mutations TP53 can carry a large number of online resources are available that contain information on TP53 mutations, domain containing the mutated residue, approximate loss of function, and possible association with cancer types. Some of the most well-known include the IARC TP53 mutation database, the p53 Knowledgebase, the TP53 Web Site, and the Database of germline p53 mutations.



Figures are made using Biorender. Zeenat Farooq is highly acknowledged for fine editing.


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Written By

Zeenat Farooq, Shahnawaz Wani, Vijay Avin Balaji Ragunathrao, Rakesh Kochhar and Mumtaz Anwar

Submitted: 15 February 2022 Reviewed: 22 April 2022 Published: 01 August 2022