p53 from pathway to therapy

Professor Sir David Lane

Department of Surgery and Molecular Oncology, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, UK

Email: d.p.lane{at}dundee.ac.uk

Abstract

In the 25 years since its first description the p53 protein has been shown to play a key role in both tumour suppression and aging. New discoveries about the function and control of p53 continue to emerge every month and attempts to exploit the system to develop better therapeutics and diagnostics are beginning to be successful in the clinic (1,2).

The genetics of the p53 pathway

The current model for p53 function as a tumour suppressor is that the protein acts as a highly regulated transcription factor. Normally p53 is a very unstable protein and is present only in minute concentrations in the cell. Even these small amounts of p53 are not fully active as a transcription factor, because the negative regulator protein, Mdm2, binds them. When cells are exposed to a wide variety of aberrant growth signals, the p53 protein is activated and stabilized and triggers the expression of downstream genes such as p21 that trigger cell cycle arrest, or Puma that triggers apoptosis. This response is potentially lethal since mice that have p53 but lack Mdm2 die very early in development at the pre-implantation stage (3,4). The response is vital to control tumour growth as mice that lack p53 develop spontaneous early onset malignancies (5). More recently however another aspect of p53 regulation has emerged. In two mouse models where p53 activity is modulated and increased by the expression of an N-terminally truncated form of the protein, accelerated aging phenotypes are seen (68). Thus, the p53 response needs to be very finely controlled in order to provide adequate tumour suppression without promoting accelerated aging.

Haploinsufficiency
Genetic and biochemical studies have demonstrated a rich variety of genes and pathways that act to control p53 function and thresholds. These must act in concert to precisely control the activity of p53. These pathways are nearly always disrupted in cancer cells that need to avoid the full p53 response if they are to grow and survive. Genetic studies have helped to clarify the importance of key players in this pathway and also suggested suitable points for therapeutic intervention. The huge number of biochemical and cell biological studies on the p53 protein can be hard to evaluate but the genetic models provide an increasingly firm basis on which to build a core understanding of the p53 pathway. Studies of the responses of heterozygote wild-type over null mice (p53+/–) have demonstrated unequivocally that the p53 locus does not obey Knudsen's two hit hypothesis. Instead there is clear evidence of haploinsufficiency at the locus. Thus, cells that contain a single copy of the p53 gene as opposed to two copies show an impaired response to radiation-induced apoptosis and tumours that arise in these mice quite frequently retain the wild-type allele (9). This also seems to occur in the tumours of individuals with Li-Fraumeni syndrome where not all tumours show loss of the single wild-type parental allele. This implies that the regulation of the p53 system is exceptionally tightly controlled and that gene dosage and transcription is a critical method by which the p53 threshold is set.

Implications of a hypomorphic Mdm2 allele
Further support for the tight control of the p53 system emerges from elegant studies on a mouse model that carries a hypomorphic allele of the Mdm2 gene (1012). The Mdm2 protein binds to p53 and targets it for degradation by acting as a ubiquitin E3 ligase that results in the specific ubiquitination of p53. In mice that are +/– at the mdm2 locus no phenotype is evident but in mice that express a slightly lower level of Mdm2 from a single hypomorphic allele thymic ablation, poor weight gain and bone marrow and intestinal cell loss are evident. All of these phenotypes are dependant on wild-type p53 and do not require any exogenous signal. The data strongly suggest that p53 is always active in these target tissues and that Mdm2 is a rate limiting negative regulator normally expressed at roughly three times the level needed to repress p53 function. The fact that the inactivation of the Mdm2 related gene Mdmx (or Mdm4) (13) also gives a p53 dependant early embryonic lethal phenotype has implied that this less well studied gene also must be critical in controlling p53 and that it cannot be compensated for by Mdm2.

The p19Arf locus and other regulators of p53
Other genes that control p53 function have also been verified by genetic analysis and the upstream genes ATM and CHK2 clearly act to regulate p53 response, as does the negative regulator of Mdm2 p19Arf. Fascinatingly while p19Arf acts as a critical activator of p53 that suppresses tumour genesis it does not alter the phenotype of the Mdm2 hypomorphic mice (11). This implies that the normal regulation of p53 is not modulated by p19Arf but that the p19Arf control pathway is activated by early events in the tumorigenic process. Other genes that modulate wild-type p53 function are fragments of p53 and mutant p53 s. Most point mutations in p53 that inactivate its function can also act as dominant negative mutants and are thus seen to act as oncogenes in transgenic mouse models. The affect of the two N-terminal truncations that promote aging is subtler however as they seem to activate wild-type function but perhaps act selectively to promote p53 function at some but not all p53 responsive promoters. Finally, the two p53 related genes p63 and p73 could also modulate p53 responses in vivo (14). Recently, further study of the ubiquitin pathway has identified two new E3 s for p53 the Cop1 (15) and Pirh2 (16) proteins. Thus, negative regulation of p53 seems to occur through at least four E3 or E3-like proteins (COP1, Pirh2, Mdm2, Mdm4) and three of these, Cop1, Pirh2 and Mdm2, are p53-induced genes. This creates a very complex and responsive control circuit able to fine-tune the p53 response. Another new gene that also looks as though it may be a powerful modulator of the response is the deubiquitinating enzyme HAUSP (1719). Again genetic studies using knock in mice have established that p53 must have an intact N-terminal transactivation domain to function as a tumour suppressor as the knock in mouse that expresses a p53 mutant at amino acids 25 and 26 (20) is no longer able to suppress tumorigenesis and in fact, despite expressing high levels of the mutant p53 protein, has an identical phenotype to a p53 null mouse. While a very large number of genes have been identified as potential down stream targets of p53 activation or suppression only three have been established as being of critical importance in vivo these are the pro-apoptotic genes Noxa and Puma (21,22) and the cell cycle regulatory CDK inhibitor p21. While knock out of these downstream response genes has a clear affect on the phenotype of the p53 dependant DNA damage response none of the knock out mice have the dramatic tumour prone phenotype of the p53 null mice.

The increased understanding of the control of the p53 pathway that derives from these genetic studies has raised many points at which the pathway may be modulated or inactivated and the pharmaceutical manipulation of the p53 pathway is within our grasp. A more sobering reflection however derives from consideration of the probable affects of functional polymorphisms at these loci in the human population. Thus, we can expect both coding and non-coding polymorphisms at these multiple control loci to be segregating in the population. This means that one can readily imagine gene combinations that will give reduced tumour protection or accelerated aging phenotypes.

Inactivation of p53 in human tumours

Mutation in p53
The most common route by which the p53 pathway is inactivated in human cancers is by mutation in the coding region of the p53 gene. These mutations have been extensively catalogued and have provided a powerful database in examining mutational spectra compared with predicted environmental causes of cancer in man. The mutations occur at many sites within the gene but the most common class of mutation is a point mis-sense mutation in the central DNA binding domain of the protein. These mutant proteins can accumulate to high levels in tumour cells where they are readily detected by antibody staining. The mutations act as dominant negative mutations as the full-length mutant protein will form mixed inactive oligomers with the wild-type protein. All of the mutants seem to be defective as transcription factors, which has formed the basis for yeast based functional assay for mutant p53 detection. Structural and physical biochemical studies of the mutant proteins has helped to promote some understanding as to why mutations at so many different locations in the protein can all inactivate its function. The current model would suggest that the core DNA binding domain of p53 is only just stable at body temperature, thus very small structural alterations are sufficient to destabilize the protein completely. This is consistent with the finding that many of the mutant p53 proteins are temperature sensitive and if expressed at lower temperature can regain p53 wild-type activity. It also helps to explain how antibodies to epitopes within the folded core of the protein are exposed by many different p53 mutations (2328). As will be discussed later this property of instability may allow the development of novel mutant p53 reactivating compounds. Why should p53 be so unstable? It is reasonable to suppose that this property may allow its rapid breakdown and close regulation as it would be more difficult to switch off a very stable protein. Our knowledge of the affects of p53 mutations is still incomplete however. In some systems the mutant proteins appeared to have gained new functions beyond that of simply acting as dominant negative p53 mutants and other models make it clear that while p53 is often described as having a domain structure these domains do have a strong interconnection with each other. Thus, a small mutation in the N-terminal domain of p53 can cause the second site reversion of a temperature sensitive mutation in the DNA binding core of the protein (29). The accumulation of mutant p53 proteins in cancer cells is not yet understood. An attractive model is that the loss of p53 transcription reduces the level of the E3 ligases so that p53 accumulates. However, other mechanisms may be at play, because in a knock in mouse model that expresses a mutant p53 protein in all tissues, p53 protein accumulation seems only to occur in cancer cells (T.Jacks, personal communication).

Other routes to p53 inactivation
Half of all human tumours retain wild-type p53 so how is the tumour suppressor function of p53 bypassed in these systems? One route is by inactivation of upstream signalling pathways; thus, CHK2 mutations have been reported in breast cancers, tumours arise in patients with ATM, loss of p19Arf expression is common in many cancers and amplification and over-expression of Mdm2 may occur in up to 9% of cancers. In cervical cancers the expression of the HPV E6 protein inactivates p53 and it is probable that as more components of the p53 response pathway are uncovered that it will be possible to see many new ways that the pathway is abrogated. The role of COP1 Pirh2 and HAUSP for example has yet to be determined. Overexpression of survival signals and of anti-apototic proteins can also help tumour cells to evade p53 activity.

Therapy and the p53 pathway
The implications of the p53 pathway in the treatment of cancer have been appreciated for the last decade. Many current treatments trigger the p53 response in normal and tumour tissue and a critical question has been whether or not the p53 status of a tumour affects the chance of a response to treatment. While in animal models dramatic affects can be seen with p53 wild-type tumours responding much better to chemotherapeutic drugs, the data obtained with human material is less clear cut. This reflects the genetic complexity of human cancer, the technical difficulties of working with patient samples and the possibility that many ‘wild-type’ p53 tumours may have other defects in the pathway. More encouraging perhaps has been the discovery and development of therapies based on the understanding of the p53 pathway.

p53 gene therapy
The delivery of Wt p53 using an adenoviral expression vector has been approved in China as part of a radiotherapy/gene therapy combination for head and neck cancer and a similar ‘p53 virus’ is in a late-stage clinical trial in the USA (30). While delivery of the virus to all tumour cells is not possible, bystander affects and possibly immune phenomena may be acting to make these therapies effective. It is possible to design more active variants of p53 by introducing mutations that block the binding of the negative regulator Mdm2 or that stabilize the core structure of the protein. While these clearly offer a superior performance in model systems more work will be needed to see if they maintain the excellent safety features of the wild-type p53 virus in human trials.

Onyx 0-15
This E1B region deletion mutant of wild-type Adenovirus was first reported to replicate preferentially in cells that lacked wild-type p53 (31). This exciting concept proved more complex on closer analysis and the viruses selective replication in tumour cells seems now to depend on tumour cells over-expressing host proteins that compensate for the E1B mutant viruses deficiencies in nuclear export (32). Nevertheless some encouraging clinical responses were seen and it is to be hoped that development of this therapy, which had the potential to attack systemic disseminated disease, will continue.

Rescuing mutant p53
A number of approaches to the rescue of mutant p53 have been proposed. Screening programmes have yielded some promising early leads, some of which have shown activity in animal models but the exact mechanism of action of these compounds is disputed. Early work suggested that peptides derived from the C-terminus of p53 could activate the DNA binding function of some mutant p53 proteins (3337). Again while the mechanism of this affect has been widely discussed no complete consensus has been achieved. Strikingly however in the last year Dowdy (38) and his colleagues have shown that such a C-terminal peptide when fused to a cell transport sequence and derived from D amino acids for stability can completely cure an aggressive intraperitoneal tumour in mice.

The non-genotoxic activation of p53 for therapy by inhibiting the p53–Mdm2 interaction
Intense study of the activation of the p53 response has revealed a number of ways to turn on the p53 response without causing DNA damage. In those tumours that retain wild-type p53 such approaches may yield exciting new treatment options. Extensive analysis of the p53–Mdm2 interaction using synthetic peptide libraries, phage display and peptide scaffolds (3942) demonstrated that blocking this interaction was able to activate p53 to induce growth arrest and apoptosis. The solved crystal structure of the interaction and the peptide database has now allowed two groups to develop small molecule inhibitors of the interaction that can trigger the p53 response (43). In a very elegant work, a group of compounds called the Nutlins have been shown to activate p53 by blocking p53 binding to Mdm2. In xenograft models these lead molecules demonstrated excellent tumour growth control without toxicity. These results have greatly encouraged the Pharmaceutical Industry by validating Mdm2 inhibition as a therapeutic target and establishing that protein–protein interactions can be appropriate targets for small molecule drugs.

Other small molecule activators of the p53 response
Screening assays based on p53 reporter cells have identified many small molecules that can activate the p53 response without causing DNA damage. A particularly potent activator is the nuclear export inhibitor leptomycin B (4446). This streptomycete antibiotic binds to and inhibits the nuclear exportin protein CRM1. The inhibition of export activates p53 dependent transcription dramatically and while it must affect many other proteins as well, cell based studies have shown that much of the cell cycle and apoptotic response to leptomycin B is p53 dependent. While normal cells can recover from a leptomycin-induced p53 dependent growth arrest the compound induces apoptotic death in p53 wild-type human tumour cells. Other molecules that are non-genotoxic activators of the p53 response include the cdk inhibitor Roscovitine (47,48). The R isomer (CYC202) of this compound is currently in clinical trial. CYC202 appears to induce p53 by partially inhibiting transcription. The link between transcription and signalling to p53 is currently the focus of intense study as it may represent a unifying principal in how the p53 pathway senses cellular stress that should invoke the p53 system.

Conclusions

The p53 protein is controlled by multiple negative regulators and activators to ensure that it achieves a high therapeutic index, protecting the organism from cancer whilst not damaging too many stem cells. In order to escape the p53 pathway nearly all human cancers have either mutated the p53 gene itself or altered the sensitivity or effectiveness of the pathway. Cancer cells are exceptionally sensitive to reactivation of p53 function and therefore treatments that achieve this promise to be exceptionally effective. The use of p53 in gene therapy has already been approved and several small molecules that can activate the p53 pathway without non-specific toxicity are under development. One of these, a small molecule cdk inhibitor is in phase 11 clinical trial. Expanding knowledge of the p53 pathway means that further strategies to manipulate the p53 pathway are emerging rapidly and one can predict that the next 25 years should see our knowledge of this fascinating protein being put to extensive clinical use for the benefit of patients worldwide.

Conflict of interest statement

D.Lane is the Founder, Director and CSO of Cyclacel Limited, a company that is developing CYC202 for cancer therapy.



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Fig. 1. p53 induced genes. When p53 is active it induces three classes of genes that have been established genetically to be essential for its function. One class exemplified by the CDK inhibitor protein p21 can cause cell cycle arrest. Another class exemplified by the BH3 domain proteins Puma and Noxa promote apoptosis, while a third class of genes exemplified by Mdm2, Pirh2 and Cop1 act to negative regulate p53 activity. The activity of this group of negative regulators, is in turn, moderated by the HAUSP and Arf proteins.

 


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Fig. 2. The activity of Mdm2 as an E3 ligase is inhibited by Mdm2 itself, acting to ubiquitinate itself promoting auto-ubiquitination and degradation. This activity is counteracted by the HAUSP isopeptidase that can stabilize and activate Mdm2. Removal of HAUSP can therefore activate p53 by reducing the activity of Mdm2 as an inhibitor of p53 function.

 


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Fig. 3. Three different E3 ligases for p53 have been described; Cop1, Mdm2 and Pirh2 can all act to promote the ubiquitination and degradation of p53. The HAUSP isopeptidase can reverse these reactions.

 
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  49. D.Lane is the Founder, Director and CSO of Cyclacel Limited, a company that is developing CYC202 for cancer therapy.
accepted May 10, 2004.