Biological functions of p53 isoforms through evolution: lessons from animal and cellular models

The TP53 tumour-suppressor gene is expressed as several protein isoforms generated by different mechanisms, including use of alternative promoters, splicing sites and translational initiation sites, that are conserved through evolution and within the TP53 homologues, TP63 and TP73. Although first described in the eighties, the importance of p53 isoforms in regulating the suppressive functions of p53 has only become evident in the last 10 years, by analogy with observations that p63 and p73 isoforms appeared indispensable to fully understand the biological functions of TP63 and TP73. This review summarizes recent advances in the field of 'p53 isoforms', including new data on p63 and p73 isoforms. Details of the alternative mechanisms that produce p53 isoforms and cis- and trans-regulators identified are provided. The main focus is on their biological functions (apoptosis, cell cycle, aging and so on) in cellular and animal models, including mouse, zebrafish and Drosophila. Finally, the deregulation of p53 isoform expression in human cancers is reviewed. Based on these latest results, several developments are expected in the future: the identification of drugs modulating p53 isoform expression; the generation of animal models and the evaluation of the use of p53 isoform as biomarkers in human cancers.     

p73 and p63: Why Do We Still Need Them?

When p73 and p63 were initially described as homologues of the tumor suppressor p53, the three family members seemed almost exchangeable, raising the question why all three were retained during evolution. It later turned out that the corresponding genes, TP63 and TP73, appear phylogenetically older than TP53, and that their targeted deletion causes severe developmental defects, in contrast to a deletion of TP53. Hence, p63 and p73 are responsible for biological effects that cannot be elicited by p53 alone. Here, we provide an overview of properties ascribed to p63 and p73 that distinguish them from p53. Differences occur at the following levels: i) protein structure, especially with regard to the aminoterminal transactivation domains and the carboxyterminal portions unique to p63 and p73; ii) regulation, affecting mRNA levels, posttranslational modifications and interaction with other cellular proteins; iii) activities, resulting in the regulation of gene expression, the programming of development, and the emergence of tumors. We speculate that, during the course of evolution, p63 and p73 have first pursued a broader range of activities, whereas p53 later specialized on genome maintenance.     

Coordinated interaction of multifunctional members of the p53 family determines many key processes in multicellular organisms

For the first time, p53 was found in complex with the viral large T-antigen in cells transformed with the small DNA virus SV40. p53 cDNA was cloned in the early 1980s, and the full-length p53 gene was cloned soon afterwards. The p53 family is comprised of three genes—TP53, TP63, and TP73—each of which is expressed as a set of structurally and functionally different isoforms. All of them intensely interact with each other, forming a united functional network of proteins. The review discusses the evolution of the p53 family and the significance of all its members in embryo development, reproduction, regeneration, regulation of aging and lifespan, and defense against cancer. Special attention is paid to the role of poorly studied members of the p53 family, p63 and p73, in carcinogenesis and tumor progression. Different isoforms of these proteins might exert opposite effects on these processes.     

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.     

p63 and p73 expression in extrahepatic bile duct carcinoma and their clinical significance

p53 plays a pivotal role in the prevention of human tumor formation. p73 and p63 are new members of the p53 tumor suppressor family, which are becoming increasingly recognized as important players in human tumorigenesis. However, the roles of these proteins are not well elucidated in extrahepatic bile duct (EBD) carcinoma. We examined expressions of the p63 and p73 genes and proteins in normal biliary epithelia, biliary dysplasias, and EBD carcinomas using immunohistochemistry and RT-PCR analysis. p63 and p73 proteins were overexpressed in 26.3 and 41.0% of EBD carcinomas, respectively. p63 protein expression was more frequent in tumors with vascular invasion (P = 0.002) and distal location (P = 0.04), while p73 expression was more common in cancers with deeper tumor invasion (P = 0.04). Patients with tumors co-expressing both p63 and p73 were found to have a significantly worse overall survival rate compared to those with either p63 or p73 expression (P < 0.05) as determined in univariate and multivariate analyses. Our results strongly imply that the p53 family members have different functions in EBD carcinomas. Our data also indicate that interactions between p63 and p73 play an important role in tumorigenesis of EBD carcinoma.     

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.     

p73 in apoptosis

The TP53 tumour-suppressor gene belongs to a family that includes the two recently identified homologues TP63 and TP73. Overexpression of p73 can activate typical p53-responsive genes and induce apoptosis like p53. In addition, activation of p73 has been implicated in apoptotic cell death induced by aberrant cell proliferation and some forms of DNA-damage. These data together with the localization of TP73 on chromosome 1p36, a region frequently deleted in a variety of human cancers, led to the hypothesis that p73 has tumour suppressor activity just like p53. However, despite its proapoptotic activity in vitro, the lack of tumour-formation in p73 knock-out mice and primary human tumour data demonstrating overexpression of wild-type p73 currently argue against p73 being a classical tumour suppressor. Interestingly, in contrast to TP53, TP73 gives rise to a complex pattern of pro- and antiapoptotic p73 isoforms generated by differential splicing and alternative promoter usage. Therefore further insight into the function and regulation of these structurally and functionally diverse p73 proteins is needed to elucidate the role of TP73 for apoptosis and human tumorigenesis.     

Evaluation of p63 and p73 antibodies for cross-reactivity

The tumor suppressor p53 is commonly mutated in human cancers. However, two homologs of p53, p63 and p73, are frequently over-expressed in tumors and are associated with tumor subtypes, clinical outcomes, and responses to therapy. There are many isoforms of p53, p63, and p73 (the p53 family). Proper detection of and discrimination between the members of this tumor suppressor family in human tissues is of critical importance to cancer research and clinical care. In this study, we assessed the specificity of several commercially available and newly generated p73 antibodies, focusing on antibodies that distinguish between the TAp73 and ?Np73 isoforms by Western analysis, immunohistochemistry, and immunofluorescence. In addition, we found that the pan-p63 and pan-p73 antibodies tested cross-react with p73 and p63 respectively. The results of this study have important implications for analysis of p63 and p73 expression and co-expression in human tumors, and for potential use of these reagents in molecular diagnostics and therapeutic decision-making.     

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.     

p63 and p73: Life and Death in Squamous Cell Carcinoma

The p53 family member p63 plays an essential role in the developing epithelium, and overexpression of the &DELTA;Np63a isoform is frequently observed in human squamous cell carcinomas (SCCs). These findings have suggested that &DELTA;Np63a might function as an oncogene within squamous epithelial cells. Nevertheless, the mechanism by which &DELTA;Np63a might promote tumorigenesis remains poorly understood, and data from mouse models implies that the p63 locus might in fact function as a tumor suppressor in these same tissues. A recent study using RNA interference in human SCC-derived cell lines shows that &DELTA;Np63a mediates an essential survival function in human SCC cells by virtue of its ability to suppress the pro-apoptotic function of the related p53 family member p73. These findings support an oncogenic role for &DELTA;Np63a and they demonstrate the existence of critical physical and functional interactions between endogenous p53 family members in human cancer. Specific chemotherapeutic agents and future targeted approaches may be able to exploit this pathway to therapeutic advantage.     

AUF1 p42 isoform selectively controls both steady-state and PGE2-induced FGF9 mRNA decay

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays vital roles in many physiologic processes including embryonic development. Aberrant expression of FGF9 causes human diseases and thus it highlights the importance of controlling FGF9 expression; however, the mechanism responsible for regulation of FGF9 expression is largely unknown. Here, we show the crucial role of an AU-rich element (ARE) in FGF9 3′-untranslated region (UTR) on controlling FGF9 expression. Our data demonstrated that AUF1 binds to this ARE to regulate FGF9 mRNA stability. Overexpression of each isoform of AUF1 (p37, p40, p42 and p45) showed that only the p42 isoform reduced the steady-state FGF9 mRNA. Also, knockdown of p42^AUF1 prolonged the half-life of FGF9 mRNA. The induction of FGF9 mRNA in prostaglandin (PG) E₂-treated human endometrial stromal cells was accompanied with declined cytoplasmic AUF1. Nevertheless, ablation of AUF1 led to sustained elevation of FGF9 expression in these cells. Our study demonstrated that p42^AUF1 regulates both steady-state and PGE₂-induced FGF9 mRNA stability through ARE-mediated mRNA degradation. Since almost half of the FGF family members are ARE-containing genes, our findings also suggest that ARE-mediated mRNA decay is a common pathway to control FGFs expression, and it represents a novel RNA regulon to coordinate FGFs homeostasis in various physiological conditions.