By Michelle Lam
Central to maintaining genome stability, tumour suppressor p53 has earned its title of the ‘Guardian of the Genome’. Over half of human cancers are reported to have mutations in the gene encoding p53, TP53, thus making it an appealing target for cancer therapy (Toshinori et al., 2011). With the majority of tumour suppressors, the acquisition of mutations in their respective genes results in a truncated or unstable protein product with lack-of-function, thereby resulting in the onset of cancer. Conversely, the majority of mutations detected in TP53 results in the production of a full-length protein that gains oncogenic functions – a paradox to the usual anti-tumour effects exhibited in wild-type (WT) p53. Such mutations in p53 are known as gain-of-function (GOF) mutant p53 (Oren et al., 2010). The anti-tumour effects in GOF mutant p53 include encouraging cancer metastasis, inhibiting cell apoptosis and contributing to chemotherapy resistance (Stein et al., 2019).
p53 is a transcription factor that plays an important role in the DNA damage response; cell apoptosis, autophagy and senescence; cell differentiation; and regulation of the cell cycle (Valente et al., 2011). As with many other transcription factors, p53 has a modular protein structure. Its N-terminal consists of two transactivation domains (TADs), TAD1 and TAD2, which are involved in stimulating transcription of p53 target genes. This is done through various mechanisms, such as the recruitment of histone-modifying enzymes. The N-terminal is followed by a proline-rich region, as well as a conserved DNA-binding domain (DBD), which binds to DNA in a sequence-specific manner at motifs known as p53 response elements (Brady et al., 2010). The p53 response element consists of two DNA decamer repeats, each of which is known individually as a half-site, that are separated by 0-13 base pairs. The C-terminal of p53 comprises of an oligermisation domain, important in facilitating the formation of the proteins’ homotetramer structure, and a lysine-rich region that can also bind to DNA, though non-specifically. It is thought that this non-specific binding of the C-terminal is involved in the regulation of DNA binding of the DBD by stabilising its binding to the response elements and promoting linear diffusion of p53 on DNA (Laptenko et al., 2015).
Usually, p53 expression levels are low in a cell. However, stressors such as DNA damage and telomere erosion will stimulate the accumulation of p53 in the nucleus, where it is phosphorylated and activated to bind to DNA (Chène, 2003). An increase in p53 expression will also activate expression of the MDM2 gene. The MDM2 protein is able to bind to the TADs of p53, with the resulting p53-MDM2 complex able to inhibit transcriptional activation of the p53 target genes. In this way, a tightly regulated negative feedback loop is formed that maintains p53 expression at a level that ensures an appropriate rate of cell proliferation and apoptosis, and to trigger the DNA repair pathways only when necessary (Liu et al., 2019). The negative feedback loop is lost with mutant p53, since it is unable to bind to DNA at the p53 response elements and induce transcription of MDM2. As a consequence, mutant p53 tends to accumulate in cancerous and damaged cells (Sobhani et al., 2020).
The majority of cancer-associated TP53 mutations are missense mutations occurring in the DBD, with many of these mutations located at six particular codons or ‘hotspots’ in the gene: R175, G245, R248, R249, R273, and R282 (Roszkowska et al., 2020). These mutations can occur directly at the residues within the DBD, known as contact mutants, or result in a change in conformation of the DBD, known as conformational mutants. Both types of mutations result in impaired binding of the DBD to the p53 response elements. Furthermore, mutant p53 has also been shown to associate with chromatin, leading to epigenetic changes that can result in oncogenesis (Pfister et al., 2017). Many of the effects observed in GOF mutant p53 are thought to be attributed to its ability to bind to and inhibit the p53-related proteins, p63 and p73, thus preventing transcription of these proteins’ target genes (Oren et al., 2010). p63 and p73 are other tumour suppressors that are in the same family as p53 and exist in multiple isoforms due to alternative splicing. The full-length isoforms of p63 and p73 each contain a DBD with 60% and 63% sequence homology with the DBD in p53, respectively. Therefore, p63 and p73 are also able to activate certain p53 target genes, thus all three tumour suppressors have shared functions, including cell cycle arrest and apoptosis. In this way, mutant p53-mediated inhibition of p63 and p73 can result in tumorigenesis (Dötsch et al., 2010).
Another way in which mutant p53 can induce tumorigenesis is through its interaction with NF-Y, a transcription factor that binds to CCATT motifs present in 30% of promoters in humans. Known target genes of NF-Y include: E2F1, cyclin-dependent kinase (cdk) 1, and cyclin A. Di Agostino et al. demonstrated that mutant p53 was able to interact with NF-Y, resulting in aberrant expression of the cyclin/cdk1 complex (Di Agostino et al., 2006). Activated cdk1 has a role in initiating S-phase of the cell-cycle and subsequent repression of origin re-licencing, important in avoiding DNA re-replication before S-phase is completed (Diril et al., 2012). DNA re-replication often generates double stranded breaks in the DNA, either by the collapse of stalled replication forks or collision of new re-replication forks with existing replication forks (Truong et al., 2011). Hence, aberrant expression of cyclin/cdk1 can lead to the replication of damaged DNA before it has been repaired, causing genome instability and potentially the onset of cancer.
Due to its prevalence in human tumours, p53 is an important target for cancer therapy. In later stages of cancer, cells often only express the mutant form of p53 and exhibit complete loss of WT p53. Hence, methods to restore WT p53 expression and eradicate mutant p53 have been deployed to treat many cancers. This can be done through chemotherapeutic interventions, with CP-31398 being the first of which was discovered and approved (Parrales et al., 2015). Moreover, another role of mutant p53 is as a potential antigen in the adaptive immune response, a novel role independent of its transcriptional enhancing abilities. It is not surprising, given that the immune system recognises cancerous cells and the cardinal role that p53 has in maintaining genome stability. The accumulation of mutant p53 in cancerous cells means that the protein indirectly serves as an antigen, activating the adaptive immune system to produce antibodies that targets the mutant p53 antigen and kills these cells. Thus, mutant p53 has been proposed as a novel target for cancer immunotherapy (Sobhani et al., 2020). Some promising research aimed at investigating this has been carried out. For example, a study by Kunizaki et al. showed that patients with oesophageal squamous cell carcinoma who had elevated S-p53Ab and SCC-Ag antibody levels had a significantly increased survival rate, demonstrating the powerful potential that this immunotherapy could have (Kunizaki et al., 2016). However, the efficacy of this treatment seems to vary amongst different cancers, and it is possible that these antibodies could also target normal cells with WT p53. More research is needed to investigate which tumour types this could be successful against and its specificity to cancer cells (Sobhani et al., 2020).
There is a plethora of ways in which WT and mutant p53 are implicated in tumorigenesis, with the former serving as an important tumour suppressor, and the latter having the potential to acquire mutations that allow it to become oncogenic itself. With emerging evidence of previously unknown roles for p53 and signalling pathways in which p53 may be implicated in, this provides the opportunity to design new anti-cancer therapies that could be extremely beneficial in ensuring that the Guardian of the Genome is able to undertake its job in protecting the human genome.
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