Role of the newer p53 family proteins in malignancy

The most recently identified members of the p53 family, p63 and p73, share certain structural and functional similarities with p53. Both p63 and p73 can bind to canonical p53-DNA-binding sites, transactivate the promoters of known p53 target genes and induce apoptosis. Despite these similarities there are many important differences. In contrast to p53, p63 and p73 give rise to multiple distinct protein isoforms that have different functional properties. Upstream signaling pathways involved in the activation of p63 and p73 differ from those involved in p53 activation. Only a subset of the DNA damaging agents that induce p53 can induce p73. Cellular and viral oncoproteins can discriminate between p53 and the newer family members. In addition, the levels of p63 and p73 are affected by certain states of cellular differentiation. Finally, it is becoming clear that the newest members of the p53 family are not classical tumor suppressor genes. In contrast to the high prevalence of p53 mutations in human cancers, p63 and p73 mutations are rare. Indeed, levels of p73 increase during malignant progression. In addition, unlike p53-/- mice, mice lacking p63 and p73 do not develop tumors, but instead have significant developmental abnormalities. Mutations in p63 have also been detected in humans with the ectodermal dysplastic syndrome EEC. Further studies are required to determine whether qualitative or quantitative differences in the expression of p63 and p73 isoforms are important in the development of human cancers.     

The p53 family: same response, different signals?

TP53, the gene that encodes p53, is a well-defined tumor suppressor gene that is frequently mutated in human cancers. Recently, two proteins homologous to p53, termed p73 and p63, were identified. Current data indicate that both p73 and p63, like p53, can induce cell-cycle arrest and apoptosis, suggesting that they might also be tumor suppressors. However, the physiological signals that can regulate p53, for example, DNA damage, have no effect on p73, as tested in several cell lines. Furthermore, the signaling pathways by which p73 (and possibly p63) induces cell-cycle arrest and apoptosis appear to be similar to those of p53, but also have important differences. Thus, the p53 family proteins are closely related but might have distinct physiological functions.     

p63 and p73: roles in development and tumor formation

The tumor suppressor p53 is critically important in the cellular damage response and is the founding member of a family of proteins. All three genes regulate cell cycle and apoptosis after DNA damage. However, despite a remarkable structural and partly functional similarity among p53, p63, and p73, mouse knockout studies revealed an unexpected functional diversity among them. p63 and p73 knockouts exhibit severe developmental abnormalities but no increased cancer susceptibility, whereas this picture is reversed for p53 knockouts. Neither p63 nor p73 is the target of inactivating mutations in human cancers. Genomic organization is more complex in p63 and p73, largely the result of an alternative internal promoter generating NH2-terminally deleted dominant-negative proteins that engage in inhibitory circuits within the family. Deregulated dominant-negative p73 isoforms might play an active oncogenic role in some human cancers. Moreover, COOH-terminal extensions specific for p63 and p73 enable further unique protein-protein interactions with regulatory pathways involved in development, differentiation, proliferation, and damage response. Thus, p53 family proteins take on functions within a wide biological spectrum stretching from development (p63 and p73), DNA damage response via apoptosis and cell cycle arrest (p53, TAp63, and TAp73), chemosensitivity of tumors (p53 and TAp73), and immortalization and oncogenesis (DeltaNp73).     

Interrelations between p73 and p53: a model, neuroblastoma

Homologies in sequence and gene organization of p53 and their relatives, p73 and p63, suggest similar biological functions. However differences exist between the p53 family members. Indeed in human tumors p53 is often mutated while p63 and p73 are very rarely mutated. In addition, in contrast to p53 which is transcribed in a unique mRNA species spanning all gene exons, each homologue expresses two types of isoforms: some with transactivation domain (TAD) showing tumor suppressive properties, the others deprived of TAD, with oncogenic properties. If p53 responds to immediate genotoxic stress, its homologues participate to the cell homeostasis of specific tissues along their development and differentiation, neuronal tissue for p73, epithelial for p63. However a collaboration between the three p53 family members has been shown to occur in response to cell genotoxic damages. Neuroblastic tumors characterized by a large spectrum of neuronal differentiation constitute a good model to study relationship between p73 and p53 as well as the regulation of their respective expression.     

The p53R172H Mutant Does Not Enhance Hepatocellular Carcinoma Development and Progression

Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53^R175H (corresponding to p53^R172H in mice) is a hotspot for mutation that demonstrates “prometastatic” gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53^R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53^R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53^flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53^R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 null counterparts, p53^R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 null HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53^R172H in mutant expressing cells, and by alternate mechanisms in p53 null cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 null HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of ΔNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.     

p73 and p63 are homotetramers capable of weak heterotypic interactions with each other but not with p53.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human cancers. Recent identification of two human homologues of p53 has raised the prospect of functional interactions between family members via a conserved oligomerization domain. Here we report in vitro and in vivo analysis of homo- and hetero-oligomerization of p53 and its homologues, p63 and p73. The oligomerization domains of p63 and p73 can independently fold into stable homotetramers, as previously observed for p53. However, the oligomerization domain of p53 does not associate with that of either p73 or p63, even when p53 is in 15-fold excess. On the other hand, the oligomerization domains of p63 and p73 are able to weakly associate with one another in vitro. In vivo co-transfection assays of the ability of p53 and its homologues to activate reporter genes showed that a DNA-binding mutant of p53 was not able to act in a dominant negative manner over wild-type p73 or p63 but that a p73 mutant could inhibit the activity of wild-type p63. These data suggest that mutant p53 in cancer cells will not interact with endogenous or exogenous p63 or p73 via their respective oligomerization domains. It also establishes that the multiple isoforms of p63 as well as those of p73 are capable of interacting via their common oligomerization domain.     

Regulating the genome surveillance system: miRNAs and the p53 super family

The p53 gene super family consists of three members; TP53, TP63 and TP73, encoding proteins p53, p63 and p73. Whilst p63 appears to have an essential role in embryonic development with a less clear role in carcinogenesis, irregularities in p53 and p73 signalling are implicated in tumour formation. As such, p53 is a tumour suppressor which is mutated in over 50% cancers and p73 was recently formally classified as a tumour suppressor based on data showing p73 deficient mice generate spontaneous tumours similar to those observed in p53 null mice. Dysregulation of both p53 and p73 has been correlated with cancer progression in many cell types and although mutation of these genes is often observed, some form of p53/p73 deregulation likely occurs in all tumour cells. The discovery that complementary micro RNAs (miRNAs) are able to target both of these genes provides a potential new means of perturbing p53/p73 signalling networks in cancer cells. Here we summarise the current literature regarding the involvement of miRNAs in the modulation of p53 family proteins and cancer development and detail the use of in silico methods to reveal key miRNA targets.