Abstract
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.
Keywords
- 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
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
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).
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 [25, 102, 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
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
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
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 “
Acknowledgments
Figures are made using Biorender. Zeenat Farooq is highly acknowledged for fine editing.
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