The rise of p53
Bert Vogelstein and Carol Prives
In 1979 the discovery of p53 was reported. The gene encoding p53 (TP53) was initially believed to be an oncogene but 10 years later it was correctly characterized as a tumour suppressor, which led to a steep rise in p53 research. We now know that the protein encoded by TP53 — one of the most commonly mutated tumour suppressor genes in human cancer — regulates many important biological activities and is itself regulated through post-translational modifications that are induced by sensors of cell stress. This Poster highlights some of the key discoveries from the past 30 years that have led to our current understanding of p53 biology and complements the Nature Reviews Cancer Focus on p53 – 30 years on, which is comprised of articles that reflect the history and the emerging directions of p53 in cancer research.
The Poster is freely available thanks to support from Roche Molecular Systems, Inc, and Genentech, Inc.
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1979-1989
High levels of mutant p53 were detected in human intestinal tumour cells (brown) but not normal cells by immunohistochemistry.
Image kindly provided by Varda Rotter and Ran Brosh.
Discovery of p53
In 1979 six groups of investigators independently reported the discovery of a ~53 kDa protein that was present in human and mouse cells. Five of these studies showed that this protein bound to the large T-antigen of SV40 in cells infected with this virus, and the sixth found it expressed in several types of mouse tumour cells. Various investigations carried out on this protein (p53), and later on the gene (TP53), suggested that it was an oncogene. This interpretation, which reflected the research climate of that time, was based on apparently compelling experimental evidence: the p53 protein was bound to the major oncogenic protein of SV40, which strongly suggested that it was a downstream effector of the large T-antigen pathway; high levels of p53 expression were found in many cancers; and the overexpression of an apparently wild-type TP53 gene transformed a normal cell into a cancer cell. Although there were a few experimental observations that did not fit with the idea that TP53 was an oncogene, the existence of tumour suppressor genes was hypothetical in the mid-1980s and so there was little reason to think otherwise.
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1989 onwards
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p53 has a pivotal role in human tumorigenesis
The turning point in p53 research came in 1989. The remaining TP53 allele sequenced from a tumour that had lost a portion of the p arm of chromosome 17, where TP53 resides, was found to be mutated. The point mutation (shown in the chromatogram) was not found in normal tissue samples taken from the patient. This, coupled with the frequent loss of 17p in tumours, indicated that TP53 was a tumour suppressor gene. It became clear that the ‘wild-type’ TP53 gene used to demonstrate that p53 was oncogenic in cell transformation experiments was, in fact, mutated. That p53 is a bona fide tumour suppressor was confirmed in 1990 by the finding that patients with Li–Fraumeni syndrome — which predisposes to diverse tumour types — had inherited TP53 mutations, and further confirmed in 1992 by experiments showing that Trp53 (which encodes mouse p53) knockout mice are prone to tumours.
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Mutations in green affect DNA contacts, those in red cause local distortions and those in purple cause global denaturation. The x-axis indicates amino acid number.
Subsequent studies have demonstrated that TP53 is more frequently mutated in human tumours than any other gene in the genome, and >25,000 TP53 mutations have been reported to date. Of these mutations, 75% occur as missense mutations that predominantly occur in the DBD. The figure shows the distribution of tumour associated missense mutations relative to the domains of p53 and highlights the six most common mutations. These mutations can affect the structure of p53 (distorting or unfolding the native conformation) and they can impair DNA binding. Experimental models have revealed that such tumour-derived mutations can have dominant-negative properties and, in some cases, can confer gain-of-oncogenic function properties in the absence of wild-type p53.
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1991 onwards
Cellular stress or DNA tumour virus infection triggers mediating proteins to activate p53 by phosphorylating it or inhibiting its ubiquitylation by MDM2. Following further post-translational modifications, the p53 tetramer regulates transcription by binding to p53 response elements and recruiting cofactors, and the protein products lead to cell cycle arrest or apoptosis.
The biological effects of p53
The first clue into the outcome of activating p53 was revealed when it was shown that the expression of wild-type p53 arrests the cell cycle in a subset of cell lines. It was subsequently shown that the expression of wild-type p53 in other cells results in cell death rather than arrest. These findings were major stimulants to the developing field of apoptosis as they showed that the regulation of apoptosis and the regulation of cell proliferation were equally important to tumour suppression. In the early 1990s two seminal observations were made: that p53 has a transactivation domain, and that it can bind to specific DNA sequences. The crystal structure of the p53 core domain bound to DNA showed exactly which p53 residues make contact with DNA and revealed how different tumourassociated mutations abrogate this binding. It is now understood that wild-type p53 functions as a tetrameric transcription factor that regulates net cell growth, inhibiting the cell cycle in some circumstances and promoting apoptosis in others, through the activation or repression of key target genes. It has also been suggested that p53 promotes apoptosis in the cytoplasm through mechanisms that do not involve transcription. Both apoptosis and cell cycle arrest have since been shown to be important for limiting the propagation of mutations following many types of cellular stress, especially DNA damage. Moreover, p53-regulated genes that function in other cellular processes, such as senescence, metabolism, autophagy, angiogenesis and DNA repair, have also been identified and these might have a role in its tumour suppressive activities (not shown in the figure).
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1993 onwards
Specific residues are modified as shown, with phosphorylation (P) in orange, acetylation (A) in green, ubiquitylation (Ub) in dark blue, neddylation (N) in pink, methylation (M) in blue and sumoylation (SU) in yellow.
The p53 regulatory network
It was known as early as 1981 that in normal, unstressed cells the level of p53 protein is low owing to its rapid turnover, whereas high levels of p53 are observed in tumours. In 1993 MDM2 was found to be a transcriptional target of p53 and was subsequently shown to be an E3 ubiquitin ligase that targets p53 for degradation, defining a negative feedback loop. Then, in 2001 MDM4, an MDM2 homologue that was first identified in 1996, was also shown to be important for the regulation of p53 activity, although its precise role remains unclear. In addition to ubiquitylation by MDM2, p53 is subject to many other post-translational modifications in response to stress, as shown in the figure. These modifications modulate the activity of p53 and its binding partners and are thought to determine the resulting p53-dependent cellular response.
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2000 onwards
Translating p53 research to the clinic
Much remains unknown about this multi-talented tumour suppressor. For example, which changes in the microenvironment favour the selection of cells with TP53 mutations? Does continuous or unrepairable DNA damage or the presence of reactive oxygen species, perhaps in association with alternating cycles of hypoxia and normoxia, influence the survival of cells with TP53 mutations? We also do not understand why the expression of wild-type p53 results in apoptosis in some cells and cell cycle arrest in others, or how the various p53 post-translational modifications might regulate this switch. Finally, and perhaps most importantly, we do not yet know how to use our knowledge of p53 for therapeutic purposes. Approaches to reactivate mutant p53 or remove inhibition of wild-type p53 are being developed and some show great promise (see the TABLE). However, the field is wide open to new, creative approaches that effectively translate p53 to the clinic. p53 research lives on.
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