The changing face of p53 in head and neck cancer

Int. J. Oral Maxillofac. Surg. 2007; 36: 1123–1138 doi:10.1016/j.ijom.2007.06.006, available online at http://www.sciencedirect.com

Invited Review Paper Head and Neck Oncology

The changing face of p53 in head and neck cancer M. Partridge, D. E. Costea, X. Huang: The changing face of p53 in head and neck cancer. Int. J. Oral Maxillofac. Surg. 2007; 36: 1123–1138. Crown Copyright # 2007 Published by Elsevier Ltd on behalf of International Association of Oral and Maxillofacial Surgeons. All rights reserved. Abstract. p53 plays a sentinel role in the pathways that prevent development of cancer by inducing apoptosis, DNA repair and cell-cycle arrest in response to different types of cellular stress. The majority of head and neck tumours harbour mutations affecting the p53 gene, and those tumours that seemingly have wild-type p53 protein most probably lack a functional p53 response as a result of mutations affecting other genes that function in the same pathways as p53. This report provides an up-to-date overview of what is known about how p53 exerts its effects. We also summarize what is known about the other p53 family members, p63 and p73, and show how they act together to influence the response to treatment. No other commonly occurring signature mutation has emerged for this tumour type, and this means that the p53 family has emerged as the frontrunner in terms of providing molecular targets that can provide new diagnostic, prognostic and therapeutic approaches.

What do we know about the common p53 gene mutations?

The p53 gene has been studied intensively since it was established that more than half of all head and neck tumours harbour p53 gene mutations, and there are now 2814 papers linking p53 with head and neck squamous cell cancer. The activity of the gene is altered by mutation or deletion, methylation or sequestration to the cytoplasm and inactivation by binding to other proteins such as human double minute 2 homologue (hdm2). The most common mutations affecting the p53 gene in head and neck tumours are missense mutations, where the wrong nucleotide is inserted into the DNA strand, 0901-5027/1201123 + 016 $30.00/0

changing the codon for a given amino acid. Typically this results in the presence of a full-length but non-functional protein. Other mutations are termed frame shifts, where there is a deletion or insertion of one or two nucleotides that changes the normal reading frame, so that a non-functional protein is produced. The other common aberration is the presence of a mutation leading to a stop codon that leads to synthesis of a truncated protein. These mutations may be homozygous or heterozygous, but if only one allele is mutated the second allele is frequently lost as a result of deletion of part of chromosome 17p69–72. The percentage of tumours reported to harbour p53 mutations, 50–70%, depends

M. Partridge1, D. E. Costea2, X. Huang1 1 Kings College Hospital Maxillofacial Unit, Kings College London, Denmark Hill, London SE5 8RX, UK; 2Department of Odontology, Oral Pathology and Forensic Odontology, University of Bergen, The Gade Institute, Haukeland University Hospital, N-5021 Bergen, Norway

Accepted for publication 29 June 2007 Available online 17 September 2007

on the method used and number of exons of the gene that are screened2,8,28,29,46,74. The majority of studies have been based on analysis of genomic DNA, but sequencing of cDNA (prepared from RNA transcribed from exons, but not the noncoding introns) means that larger fragments of the gene can be analysed using automated methods, reducing the cost and time taken to establish whether a mutation is present. Investigations incorporating this approach to assess the entire length of the gene have suggested that a higher percentage of tumours harbour p53 gene mutations3,49,81,98. When cDNA sequencing is performed, strict precautions must be taken to avoid contamination. It seems likely that the literature contains some

Crown Copyright # 2007 Published by Elsevier Ltd on behalf of International Association of Oral and Maxillofacial Surgeons. All rights reserved.

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reports that include false-positive results, as unusual mutations were detected, or there were discrepancies when both genomic DNA and cDNA were examined, or several cases in a small series appear to harbour the same mutation. When these studies are excluded the percentage of cases harbouring these mutations is in line with the original reports. The spectrum of mutations in head and neck cancers is broadly similar to that found for other squamous tumours. Most studies have examined exons 5–8, 5–9 or 4–9 of the gene, with those that have included additional exons finding a small increase in the number of mutations, such that it is now accepted that examination of exons 4–9 provides a comprehensive screen for mutations in head and neck cancer patients. Codons 238–248 (exon 7) and 278–281 (exon 8) are recognized as hotspots for mutations in these patients. These hotspots are localized at the DNA-binding domain and prevent p53 binding to the promotor region of target genes. For example, p53 exerts its apoptotic effects by combining with the apoptosis-stimulating protein of p53 (ASPP, see below) but mutations affecting codons 248 and 273 prevent this interaction, blocking the activation of this pro-apoptotic protein. In addition to these effects, mutant p53 can markedly reduce the binding of wild-type p53 to the p53 response element in the promoter of many p53 target genes, including p21, and in this way the mutant protein is said to have a dominant-negative effect over the wildtype counterpart. A number of so-called gain-of-function p53 mutations are also recognized. These have activities that are not associated with the wild-type protein. For example, they can exert mitogenic effects by stimulating production of growth factors, including basic fibroblast growth factor, interleukin 6, vascular endothelial growth factor or expression of the receptors for epidermal growth factor or insulin-like growth factors. Some mutants also up-regulate c-myc expression, or are able to enhance the activation of topoisomerase I, an enzyme that causes breaks in single-stranded DNA and plays a role in DNA ligation leading to genetic instability. There is good evidence that specific signature mutations affecting the p53 gene are associated with exposure to known carcinogens. For example, ultra-violet light induces cytosine–thymidine (C-T) base changes and CC-TT tandem transitions48. These mutations are commonly associated with cancer of the lower lip.

The spectrum of mutations that occur most frequently in head and neck tumours, and also in squamous cell carcinoma of the lung, are at guanine (G) nucleotides and associated with the carcinogens that are present in tobacco smoke9. Further research has revealed that treatment of cells with benzo[a]pyrene induces mutation of the p53 gene most frequently at codons 157, 248 and 27318, and the majority of mutations occurring at these three codons were G-T transversions. These codons are the sites of common mutations in the p53 gene in lung cancer, providing a direct link between a carcinogen present in cigarette smoke and the mutations that occur in human cancers. Other ways of inactivating p53

Several studies have shown that tumours that seemingly harbour wild-type p53 may not have functional p53, as this protein is activated by binding of the human papilloma virus 16 (HPV16). This virus may be associated with the development of a subset of tumours arising in non-smokers28,34,45. The viral E6 protein of HPV16 competes with hdm2 for binding to p53 and can recruit a cellular ubiquitinligase to p53, which is then ubiquitinated and targeted for degradation through the proteasome54. When tumours harbour HPV16, p53 mutations are not detected. There is no selection pressure to expand clones of precursor cells that harbour mutant p53, as the function of this protein has already been lost17,28,31,95,100. p53 may also be inactivated by overexpression of hdm2 in some tumour types, although there is scant evidence that this mechanism plays a role in the molecular pathogenesis of head and neck cancers73. The p14 tumour suppressor protein, the alternative product of the INK4 (p16) gene, can bind to the hdm2 protein preventing the association of hdm2 and p53, thereby releasing p53 from the complex. Over time it has also become apparent that the p53 pathway is not functional in almost all tumours, as most proteins that are regulated by p53, including p21, Bax, Gadd45 and thrombospondin, show abnormal patterns of expression in head and neck cancers. The p53 mutation database

The p53 mutation database (http:// p53.free.fr/) shows that more than 1500 mutations have been reported in 15,000 tumours from diverse sites, and that the 11 hotspots for mutation in the DNAbinding domain account for more than

80% of all mutations. Some of the mutations in the database have only been reported once and a second database has been created to describe the functions of the various mutant proteins. At present, this covers approximately 10% of the p53 mutations recorded, and has revealed that the very many different mutations have distinct functions in the cell and different abilities to transform cells. Comparison of the two databases showed that infrequent mutations are associated with almost normal p53 protein activity, and the finding of multiple mutations in one tumour, silent mutations or absence of mutations at the recognized hotspots was frequently associated with approaches incorporating nested (two rounds of) polymerase chain reaction for which the controls were not satisfactory. SOUSSI et al.91 showed that these ‘aberrant’ mutations, that differ significantly from the profile observed in other studies, can have a profound effect on any analysis of the p53 mutation database. This led to the recognition of a requirement to assess the quality of data accepted for inclusion in the p53 databases, and the establishment of a curator committee of p53 specialists who will propose guidelines to improve the reliability of the data collected. Initially the ‘clean up’ will be for the lungspecific p53 database, but the aim is to extend this to include all tumour sites in due course. What does the p53 gene do?

The function of p53 is to integrate signals emerging from a wide range of cellular stresses, by inducing adaptive and protective cellular responses by activating (transcribing) specific sets of genes. The bestcharacterized response to stress is the cellular response to DNA damage. Other cellular stresses include the presence of activated oncogenes, oxidative damage, viral oncogenic proteins (for example, SV40T, E1A), hypoxia, abundance of cytokines and growth factors, and other agents that damage DNA such as radiation and UV light (Fig. 1). The p53 protein allows cells to respond to these stresses as it acts as a transcription factor, or switch, that regulates the expression of a broad range of target genes. It is essential for preventing inappropriate cell proliferation, by inducing growth arrest at different points of the cell cycle and maintaining the integrity of the genome following DNA damage or inducing apoptosis, making this protein the ‘guardian of the genome’50.

The changing face of p53 in head and neck cancer

Fig. 1. Summary of the key interactions of the p53 protein. Table 1. Genes that are activated by p53 Cell-cycle arrest p21, 14-3-3-s, cyclin G, proliferating cell nuclear antigen (PCNA), Gadd45, topoisomerase DNA damage sensing and repair Gadd45, ERCC2 (XPD), TF11H Apoptosis Bax, Apaf 1, Puma, Noxa, Fas, PIG3, p53AIP, poly ADP-ribose polymerase (PARP) Angiogenesis Thrombospondin

The structure of the p53 gene and protein

This gene is located at the short arm of chromosome 17 and has a complex structure with 11 exons of which 10 are coding. The p53 protein is comprised of 393 amino acids and divided into five domains with each domain having a different structure and function (Fig. 2). The first N-terminal domain has 42 amino acids and is often termed the transcriptional activation (TA) domain. This interacts with elements of the transcriptional machinery required for synthesis of

Fig. 2. The structure of the p53 protein.

new mRNA. This TA domain is critical for activation of the genes that are the targets of p53 (Table 1). This region also contains the binding site for hdm2, an important regulator of p53, as when hdm2 is bound to p53 it is involved in transport of p53 out of the nucleus to the ubiquitin-mediated proteolytic machinery. p53 which is bound to hdm2 is also unable to interact with the transcriptional apparatus. The second domain (amino acids 61– 94) is a proline-rich region and mutations here reduce the ability of p53 to mediate apoptosis and cell-cycle arrest. A polymorphism at codon 72 results in either

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proline or arginine being present at this site, giving rise to two variant forms of wild-type p53 known as p53 (Pro) P and p53 (Arg) R. These proteins are functionally distinct and influence the response to radiotherapy and chemotherapy. Amino acids 97–300 comprise the central domain known as the DNA-binding domain. This includes the region responsible for sequence-specific DNA binding. This domain folds into a scaffold that serves as a docking station for residues within both the major and minor grooves of DNA so that p53 can bind with its target genes. The majority of missense p53 gene mutations occur here and prevent this interaction. Amino acids 300–393 comprise the carboxy terminal region, which contains two further domains joined by a flexible linker. Within this region is the oligomerization domain through which p53 monomers form dimers and tetramers. Several nuclear localization signals, a lysine-rich nuclear export signal, which is important when p53 is shuttled from the nucleus to the cytoplasm, and the lysine residues to which ubiquitin binds when p53 is to be degraded are also located here. The carboxy (C) terminal amino acids (363–393) form an open basic domain that interacts with the DNA-binding domain to negatively regulate the ability of p53 to bind to specific gene sequences, thus maintaining p53 protein in an inactive form. A number of post-translational events (for example, phosphorylation and acetylation) occur at the C-terminal domain and convert p53 to its active form, so that it can bind to the promoter region of its target genes. The C-terminal domain is also responsible for the ability of p53 to bind non-specifically to DNA molecules, a function that is important for DNA repair.

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Changes in p53 levels in response to stress

Regulation by controlling interaction with other proteins

In normal cells, the levels of p53 protein are very low and almost undetectable. This reflects the rapid turnover time of this protein, which is as short as 5–20 min. In contrast, when cells are proliferating rapidly, or exposed to stressful events, the levels of p53 rise dramatically. Once stabilized, p53 needs to be transported from the cytoplasm to the nucleus so it can act as an efficient transcription factor; it also forms tetrameric complexes with other p53 molecules. Once this occurs, p53 tetramers associate with the various components of the transcriptional machinery, and synthesis of mRNA that will trigger the cellular response to stress begins. Monomeric forms of p53 are able to function as a transcription factor but tetramers of four identical subunits are much more efficient40. The presence of a mutant p53 molecule in the tetramer decreases the ability of p53 to promote transcription, but at least three DNA-binding defective p53 mutations are required to inactivate the tetramer.

Rapid degradation of p53 helps to ensure that levels of this protein remain low in normal cells and, as outlined above, this is achieved by binding of hdm2 to the N terminus of the protein, blocking its ability to function as a transcription factor. Binding of hdm2 also shuttles p53 from the nucleus to the cytoplasm, a process that involves exporting and its subsequent degradation through the ubiquitin pathway. When ubiquitin chains are added to p53, the protein is degraded into peptides by the proteasome. This has two subunits, a catalytic core responsible for protease activity and a regulatory unit involved in protein–ubiquitin recognition. This activity means that the proteasome has a critical role, allowing cells to progress through the cell cycle by degrading key regulatory proteins at appropriate times, and an indirect role by regulating the levels of transcription factors such as p53. hdm2 is itself a member of a family of proteins and its homologue, hdmX, can stabilize both hdm2 and p53. The hdm2 gene has a single nucleotide polymorphism in the promoter region (either T or G), the G form of which supports enhanced binding of a transcription factor leading to elevated levels of hdm2 without gene amplification. HONG et al.37 demonstrated that individuals with the p53 Arg/Arg and hdm2 GG genotype had an increased risk of oesophageal cancer. This may be due to increased levels of hdm2 that might be expected to reduce the effectiveness of any p53-driven response. Other regulators of p53 also exert their effects via hdm2, for example the promyelocytic leukaemia protein increases the

Controlling the levels of p53

As p53 has such significant effects in cells, the levels of this protein need to be kept under strict control. Regulation of p53 occurs at several levels. These include transcription, post-translational modifications (phosphorylation, acetylation, sumoylation), interaction with other proteins and its intracellular localization.

Regulation by controlling transcription of the p53 gene

Little is known about how transcription of the p53 gene itself is regulated. Protein kinase C delta can stimulate transcription of p53 in response to DNA stress1, and hdm2 interacts with p53 to block its activity as a transcription factor.

levels of this p53-binding protein by sequestering hdm2 in the nucleolus, whereas survivin decreases the levels of hdm2. The action of the peptidyl-prolyl isomerase nucleoprotein (Pin 1) is also important, as binding of this protein to p53 changes its conformation, so that it can no longer interact with hdm2 and p53 is stabilized. Binding of Pin 1 to p53 also facilitates other post-translational modifications, particularly acetylation, and is thus an important regulator of p53 function that is frequently overexpressed in cancers. When p53 is activated after exposure to abnormal levels of growth factors or oncogenic stimuli, it is the degradation of p53 protein that is blocked. This occurs as p14 binds to hdm2 and can target this protein for degradation, thereby releasing p53. This pathway ensures that aberrant mitotic signals will trigger a compensatory inhibitory response to limit cell proliferation. p14 is not expressed by approximately 15% of head and neck cancers, and in theory this may lead indirectly to higher levels of hdm2 and increased stabilization of p53.

Regulation by controlling the intracellular location

The stability of p53 is also influenced by its intracellular localization, which changes with the different phases of the cell cycle (Fig. 3). In normal cells, p53 is predominantly located within the nucleus from G1 through the G1-S transition, but moves to the cytoplasm as the cell cycle progresses. Following exposure to stress, p53 is stabilized and rapidly relocated to the nucleus so that it can serve as a transcription factor, although p53 may also be

Regulation by controlling posttranslational modifications

When p53 is activated as a result of DNA damage, this is associated with phosphorylation and acetylation of the protein. Phosphorylation of p53 allows the protein to bind non-specifically to DNA, but when the protein is acetylated within the Cterminus this alters the configuration of the protein so that p53 can now bind very specifically to the promoter in its target genes.

Fig. 3. Cartoon summarizing the key regulators of the cell cycle.

The changing face of p53 in head and neck cancer detected in cytoplasm and in the mitochondria, as it is an important regulator of apoptosis.

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In total, more than a 100 genes are known to be activated by p53, and the list of downstream targets of p53 include those involved with cell-cycle arrest, the sensing of DNA damage, DNA repair, apoptosis and angiogenesis (Table 1 and Fig. 4). Activation of p53 also increases transcription of hdm2, thereby controlling its own activity by a negative feedback loop.

(cdk) cdk4/6 and cdk2 releases E2F from Rb, permitting transcription of genes that include cyclin E and PCNA. These amplify the signal to switch from G1 to S phase that is required for DNA replication (Fig. 4). When a p53-mediated stress response occurs, levels of p21 rise and, whereas binding of one molecule of p21 allows cdk activity, binding of two p21 molecules is inhibitory. p21 also binds to PCNA, blocking its activity as a DNA polymerase processing factor in DNA replication, effectively stopping replication, but leaving its ability to participate in DNA repair intact.

stimulated by p53 at this time include 143-3-s, which binds to the phosphorylated cdc2-cyclin B kinase and exports it from the nucleus, and Gadd45, which binds to and dissociates the cdc2-cyclin B kinase and inhibits various cdks. The effect of these genes is to block progression through this part of the cell cycle. p53 may also delay mitotic arrest through regulation of the topoisomerase II/cdc2 interaction. Activated p53 can suppress the expression of topoisomerase II, reducing the amount of complexes formed with cdc2, and inhibiting chromatin condensation and hence mitosis51.

The role of p53 in G1 arrest

The role of p53 in G2 arrest

Activated p53 may stimulate transcription of the p21 gene responsible for arrest in the G1 phase of the cell cycle. The G1-S checkpoint is principally regulated by a gene transcription complex that includes Rb and the E2F transcription factors. The hypo- or under-phosphorylated form of Rb binds to these transcription factors, blocking their ability to bind to DNA so that transcription of target genes does not occur. In contrast, phosphorylation of Rb by the cyclin-dependent kinases

p53 also plays a role in regulation of the G2-M checkpoint, preventing premature entry of cells that have just divided into another division by inducing cell-cycle arrest. This checkpoint is critically dependent on activation of cyclin B. This activation process is regulated by cell-cycle checkpoint 2 (cdc2). During G2, cdc2 is maintained in an inactive state, but as cells approach M-phase it is activated by the phosphatase cdc25, and this drives the cell into mitosis (Fig. 4). The genes that are

The role of p53 in responding to DNA damage

The target genes of p53

In response to DNA damage occurring as a result of ionizing or ultraviolet radiation, stress-induced kinases are activated. These include the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia Rad3-related (ATR) kinases that are activated in response to irradiation and ultraviolet light, respectively (Figs 4 and 5). These kinases in turn control the activation of effector kinases, the checkpoint homologue kinases (Chk1 and Chk2)

Fig. 4. The p53 network, with red arrows indicating pathways of p53 activation or stabilization, blue arrows the key targets of p53 and black arrows the pathway of degradation. indicates that a p53 target is activated by phosphorylation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Partridge et al. causes the mitochondria to swell and rupture, releasing pro-apoptotic factors cytochrome C, Smac, Omi and apoptosisinducing factor (AIF) into the cytoplasm. p53 may also repress the activity of antiapoptotic genes that include Bcl 2 and other Bcl 2 family members. p53 also has a direct pro-apoptotic role which depends on its ability to translate from the nucleus to the mitochondria, where it forms complexes with Bcl xL and Bcl 2, favouring change in mitochondrial membrane permeability and cytochrome C release. The extrinsic apoptotic pathway

Fig. 5. DNA sensors and the response to DNA damage.

and DNA-dependent protein kinase (DNA-PK). These enzymes phosphorylate serine and threonine residues in the Nterminal transactivation domain of p5319. Phosphorylation in the N-terminal domain of p53 inhibits binding of hdm2, and enhances transcription of p53 so that levels of the p53 protein rise and it is not degraded. Following DNA damage, p53 also induces transcription of genes involved in DNA sensing and repair. These include p48/DDB2, XPC and Gadd45. p53 also interacts with ERCC2 (XPD) and ERCC3 (XPB), two components of the transcriptional factor TF11H. These proteins are helicases that unwind the DNA duplex at the site where DNA repair is required. When DNA damage is beyond repair, activation of p53 leads to apoptosis via both transcription-dependent and transcription-independent mechanisms. These may act together to ensure that the cell death programme proceeds efficiently. When the damage to DNA is not severe, p53 stimulates transcription of genes that mediate a delay in the G1 and G2 phases of the cell cycle to allow for DNA repair. Activation of the checkpoint kinases also increases the levels of various downstream targets, including p73, c-abl and the E2F transcription factors, such that expression of the anti-apoptotic genes, Bcl 2, Bcl xL, bcl w and Mcl 1, is reduced. If the damage is severe, the pathways that ultimately lead to apoptosis will be activated and p53 will stimulate transcription of a different set of genes, including the pro-apoptotic genes Bax, PIG3, Puma, Noxa, Fas and poly ADP-ribose polymerase (PARP). Activation of p53 also represses survival-signal genes such as Bcl 2.

The apoptotic stimulatory p53 proteins (ASPPs) 1 and 2 also enhance the ability of p53 to bind to the promoters of proapoptotic genes, including Bax and PIG386, to stimulate apoptosis, but not to genes that cause cell-cycle arrest. A further protein that modulates these interactions has been described (inhibitor of the apoptotic stimulatory p53 protein) iASPP. This competes with ASPP1 or ASPP2 for interaction with p53, blocking its ability to serve as a transcription factor. STAT1 can also act as a co-activator of p53 to induce expression of the apoptotic stimulatory proteins Bax, Noxa and Fas93,94. It is the intrinsic apoptotic pathway that is primarily mediated via the mitochondria, that is driven by p53 with the extrinsic pathway being activated via death with caspase 8 being used to augment the apoptotic response (Fig. 4).

The p53 protein can also promote apoptosis through activation of the death receptors (extrinsic pathway) located at the cell membrane. These include Fas, TRAIL receptors, DR4 and DR588. To add to the complexity of the role of p53 in apoptosis, several other components of the apoptotic machinery, including Apaf-1, caspase 6, PTEN and the REDOX genes, can be activated by p53. p53-independent apoptosis

The cell also has p53-independent pathways through which DNA-damaging agents induce tumour cell apoptosis. One involves the tyrosine kinase c-abl, which may be activated by DNA-PK or ATM. This pathway generates the same signals as p53 via the p38 (mitogen-activated protein) MAP kinase pathways. A second p53-independent apoptotic pathway involves the transcription factor E2F that stimulates transcription of Apaf-1, leading to activation of caspase 9, induction of caspase proenzymes and transcription of p73 (Fig. 5).

The intrinsic apoptotic pathway

As outlined above, p53 induces apoptosis through the intrinsic pathway by modulating the activities of many of the proteins that control the integrity of the mitochondrial pore complex. The Bcl 2 protein is a key negative regulator of apoptosis, but this inhibition may be overcome when levels of pro-apoptotic proteins rise. p53 also up-regulates the expression of a family of proteins that modulate the levels of Bcl 2, and increases the expression of apoptosis-inducing protein (AIP) that mediates cytochrome C release from the mitochondria. When the pro-apoptotic proteins predominate, the structural changes that occur in the mitochondrial membrane lead to an increase of calcium within the cells. This

What influences whether the cell lives or dies?

Whether a cell will undergo cell-cycle arrest or apoptosis after exposure to stress is dependent on whether p21 or pro-apoptotic proteins predominate at any given time. It is not yet known exactly how a cell decides to respond to stress by a period of growth arrest in either the G1 or G2 phases of the cell cycle, or whether it will attempt to repair any DNA damage, undergo apoptosis, differentiate or become senescent. Historically it was considered that the level of p53 was the key factor influencing the response, but it is now clear that posttranslational modifications and p53 gene mutations are also important in determin-

The changing face of p53 in head and neck cancer ing the overall response. For example, phosphorylation at serine 46 favours apoptosis rather than cell-cycle arrest, and binding of the ASPPs also regulates the ability of p53 to activate targets genes that favour apoptosis. Other members of the p53 gene family, p63 and p73, are essential for p53-induced apoptosis, and almost all tumours have acquired defects in p53 family pathways that block apoptosis and prevent the cell-cycle checkpoint controls from functioning, Other p53 family members

Two other members of the p53 gene family have been identified: p63 and p73. These proteins have high sequence and structural homology to p5342,43,87,102 and seemingly similar functions, but studies using knockout mice have revealed that while p63 and p73 knockouts exhibit severe developmental abnormalities but no increased cancer susceptibility, the p53 knockouts develop normally but are more susceptible to tumour development. The p63 and p73 genes have two promoters that result in the synthesis of two classes of proteins: those containing a TA domain (TAp63 and TAp73, the long forms of these proteins) and those that do not have this domain (DNp63 and DNp73, the short forms of these proteins). The TA proteins have very similar functions to p53 in cell culture, including transactivation of many of the p53 target genes, whereas the DN proteins are inhibitory. This means that p63 and p73 can have both pro-apoptotic (TA) and antiapoptotic (DN effects). The TA and DN forms of p63 and p73 are also made more complex by multiple splicing at the carboxy terminal domains, resulting in the identification of nine transcripts for p73: a, b, g, d, e, z, h, h1 and f (a being full length), and three for p63: a, b and g61. The shorter inactive forms of p63 and p73 can compete for binding sites with full-length p53 and p63. In general, there are many similarities between the p53, TAp73 and TAp63 proteins, and between DNp73 and DNp63. For example, TAp73 has similar transcriptional activity to p53 in vitro, activating p21, 14-3-3s, Gadd45 and hdm241,103, as well as apoptosis59. The activities of p63 and p73

TAp73, like TAp53, is involved in the response to DNA damage. The pathway by which TAp73 exerts its effects involves the mismatch repair gene MLH1 and c-abl, whereas the p53 proteins exert their effects

via the stress-induced kinases ATM and ATR. Recent studies have shown that expression of p73 and its downstream targets is induced by cisplatin, UV and taxol, and as TAp73 has similar critical activities to p53, this protein has been described as the ‘assistant’ guardian of the genome60. TAp73 also has similar activity to p53 in that it stimulates transcription of Bax, Puma and Noxa, but TAp63 is relatively weak. The activity of TAp63 is less well characterized, but cells overexpressing TAp63g, TAp63a, DNp63a and DNp63g show poor or no detectable apoptosis compared to those overexpressing p53 or TAp73. Expression of p63 in normal oral mucosa

An unexpected finding was that the basal cells of mature normal human epithelium, including the epidermis and oral mucosa, strongly express p63 proteins, predominantly the DNp63 isotype (ratio is 100:1 of DNp63 to TAp63). The presence of this isotype seems to be responsible for the maintenance of the proliferative potential of basal keratinocytes in mature epithelia36. Expression of DNp63 falls as soon as these cells withdraw from the stem-cell compartment25. This observation is consistent with the role of DNp63 in binding to p21 and 14-3-3s promoters and repressing them. Thus, as keratinocytes differentiate, expression of DNp63a is lost, leading to increased expression of p21 and 14-3-3s that mediates cell-cycle arrest47. The interaction between p53, p63 and p73

Recent reports have also highlighted the fact that complex interactions between p53, p63 and p73 may occur. For example, when the function of both p63 and p73 genes is knocked out, p53 cannot trigger apoptosis on its own, even though this protein is expressed at normal levels and can be shown to be functional24. In this study, FLORES et al. demonstrated that TAp73 and TAp63 were required for p53-mediated activation of Bax, Noxa and PARP, and stimulation of apoptotic responses. Nuclear oncogenes, such as high levels of myc and HPV E7, which forms a complex with Rb, also raise the level of p73, such that it is always higher in cancer than normal cells. Wild-type p53 binds to p63 and degrades it79, and overexpression of p63 is frequently associated with head and

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neck tumours (e.g. see refs13,35,38,66,90), most likely as p53 function is lost. DNp63 can also block p73-dependent apoptosis, by binding to and blocking transcription of p73. ROCCO et al.82 suggested that these events are probably all part of the fundamental pathways that allow damaged cells to survive during the carcinogenic process. To date, no inactivating mutations have been reported for p63 or p7361. Loss of heterozygosity on chromosome 1p36, the site of the p73 gene, has been reported in some cancers, but there is no good evidence that this gene is consistently deleted in human tumours12. Clinical applications of p53 and the p53 family members How are mutations detected in tumours?

Stabilized p53 protein can be detected by immunohistochemistry, and a range of antibodies are available that can be used to examine paraffin-embedded tissues as well as frozen sections. Reagents that can recognize p53 when phosphorylated at specific sites are also available. The concentration of antibody used determines the number of lesions that are scored as positive for p53 protein expression, and this has led to the recommendation that a specific dilution of an antibody recognizing p53 termed D07 is used for optimal clinical use, with the caveat that the optimum dilution may differ slightly between laboratories due to variations in fixation conditions and antigen retrieval57. Immunohistochemistry can serve as a rapid screen for the presence of mutations, but cannot be relied upon to detect p53 gene mutations with certainty, as the concordance between the presence of p53 mutation and detection of protein by immunohistochemistry may be poor32,53. This is because levels of p53 may be elevated in the absence of a mutation, reflecting the fact that the protein may be stabilized as a consequence of ongoing DNA damage73,89, and high levels of p53 may be found in the normal oral mucosa from smokers. The correlation between immunohistochemistry and mutation detection is also influenced by the failure to pick up nonsense or splice-site mutations by immunohistochemistry, and specific p53 mutations lead to truncated proteins that are frequently not detected by immunohistochemistry. Recent studies have suggested that it may be possible to assess the function of p53 by determining the pattern of both p53 and hdm2 expression65. But hdm2 is rarely

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expressed in oral tumours73, and at the present time molecular approaches provide the best method to identify p53 mutations. Mutations affecting p53 can be detected by sequencing genomic DNA or cDNA that is reverse transcribed from exons 4– 10, as outlined above. New approaches for mutation detection based on development of p53 gene chips are in development, and may provide a more rapid method for identifying mutations or polymorphisms. Preliminary studies suggest that the sensitivity and specificity of gene chips for detecting p53 mutations are similar to DNA sequencing8. p53 and prediction of survival

It is generally considered that in the future it will be possible to use information about the spectrum of mutations present in cancer to provide a more accurate staging system than the current TNM system. The p53 gene is one of the most commonly mutated targets, and many studies have evaluated whether overexpression or mutation of p53 can be used to help gauge a patient’s prognosis (Fig. 6). Some investigations have suggested that outcome is better for patients who harbour wild-type p53 but others have failed to confirm this association77. The evidence that is emerging outlining the role of the p63 and p73 proteins suggests that relying on p53 as a prognostic marker

Fig. 6. Clinical applications of p53.

is overly simplistic, particularly as this gene pathway is nearly always knocked out by other mutations, even if the p53 protein is wild type. Many studies have failed to show a clear-cut association between p53 mutation and clinico-pathological features of the tumour or prognosis. At one time it was considered that mutations at DNAbinding sites might confer an adverse prognosis8,101, but this association has not been confirmed in larger studies. Some investigations suggest that the restricted pattern of mutations reported for laryngeal tumours may reflect the fact that these epithelia are exposed to the carcinogenic effect of tobacco, but not alcohol, whereas tumours that occur in the mouth and oropharynx are associated with a broader range of risk factors including alcohol, HPV and bacteria. p53 status and prediction of the response to radiotherapy or chemotherapy

More than half of all head and neck cancer patients receive radiotherapy at some time during their treatment, and chemoradiation is increasingly the treatment of choice for large oropharangeal tumours as well as for palliation. Irradiation causes DNA damage directly by causing single- and double-strand breaks, and indirectly via generation of free radicals, and is thus most effective at eliminating tumour cells that have a good blood supply.

At present, the majority of patients with tumour deposits in two or more nodes, or aggressive tumours that show evidence of vascular or neural invasion, or those associated with positive or close margins, receive postoperative radiotherapy. This adjuvant treatment does not prevent relapse and loco-regional recurrence remains the most common cause of treatment failure for these cases. Ionizing radiation exerts its effects by causing DNA damage and inducing apoptosis, such that it is to be anticipated that the presence of a p53 mutation might reduce the effectiveness of radiotherapy, but studies relating the presence or absence of p53 mutations to outcome following radiotherapy alone, or adjuvant radiotherapy post-surgery, show no consistent relationship. There are some small investigations which do show a positive correlation, but no consistent patterns have emerged. In a recent study, ERIKSEN et al.23 showed that p53 mutations were not related to local control or survival, but patients with tumours that harbour these aberations, may benefit from reduced overall treatment time.

How do chemotherapeutic drugs exert their actions?

The cytotoxic agents most frequently used for chemotherapy or for chemoradiation to provide palliation for head and neck cancer patients are 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum (cisplatin), although the use of taxol and paclitaxel is increasing. These agents exert their effects via a range of different mechanisms that include direct toxic effects on DNA, stimulation of mitochondrially mediated (intrinsic) and death receptor-induced (extrinsic) apoptosis and necrosis. The anti-tumour effect of 5-FU is exerted by formation of a ternary complex with thymidylate synthase, which inhibits DNA synthesis. Treatment with cisplatin results in the entry of free platinum into cells. This binds to DNA and results in the formation of inter- and intra-strand crosslinking as well as single-strand DNA breaks39. These cross-links prevent transcription and DNA replication, as the double helix of DNA must be unwound over short distances to facilitate the action of the various RNA and DNA polymerases. The intra- and inter-strand cross-links formed in DNA by cisplatin disrupt these processes and prevent transcription, and also activate the DNA sensors. This leads to apoptosis by activating

The changing face of p53 in head and neck cancer proteins that recognize damaged DNA via the intrinsic and extrinsic pathways. The cellular response to irradiation and chemotherapy

Following irradiation a cell may undergo apoptosis, permanent arrest or temporary arrest while DNA repair occurs. Immediate apoptosis occurs in some normal cell types, for example, haematopoietic precursors and in the crypts of the small intestine. Fibroblasts undergo irreversible growth arrest. If the dose of irradiation is relatively low, cells that are growth arrested at that time may attempt repair via processes termed homologous recombination and non-homologous end-joining. If this is successful they may subsequently continue to proliferate. If the damage is severe, such that repair is not possible, then the cell will undergo apoptosis. If the damage is not lethal but too great for repair, the cell will try and resume proliferation with its acquired mutations. This may result in mitotic catastrophe, an event that is typically caused by mis-segregation of chromosomes. Cells undergoing mitotic catastrophe often have micro-nuclei, and giant cell formation and multi-nucleate cells are common. Mitotic catastrophe frequently leads to apoptosis, but cells that do not undergo mitotic catastrophe on the next, or subsequent, rounds of division may continue to proliferate with damaged DNA. The aberrations that favour tumour growth also aid some treatment approaches, as the defects in checkpoint control mean that cells that are unable to repair enter mitosis and undergo mitotic catastrophe. Pathways that influence the cellular response to radiotherapy and chemotherapy

Historically, the p53 gene was considered the most important determinant of response to radiotherapy or chemotherapy. Many studies conducted during 1980– 1990 that aimed to correlate the response to treatment with p53 gene expression produced conflicting results77. As outlined above, it is now known that apoptosis is a fundamental mechanism of cell death following treatment with cytotoxic agents. Wild-type p53 may enhance chemosensitivity by promoting apoptosis, and some studies have suggested that the presence of a p53 mutation is associated with a lower response to chemotherapy11,16,26. Contradictory studies have also been reported, in that tumours that overexpressed p53 were more responsive

to cisplatin than those that did not express p536. Resistance to cisplatin can be due to increased levels of Bcl proteins, or increased expression of butyrate responsive factor 1 which increases the levels of inhibitor of apoptosis proteins52. Recent investigations have shown that some of this variation in chemosensitivity may be due to a common sequence polymorphism of the p53 gene that results in either proline or arginine at amino acid position 72. This polymorphism occurs in the prolinerich domain of p53, which is necessary for the induction of apoptosis. DUMONT et al.20 found that the Arg72 variant induces apoptosis significantly better than the Pro72 variant, most likely as this form can localize to the mitochondria more efficiently and stimulate the release of cytochrome C into the cytosol. This Arg 72 polymorphism is associated with a good response to chemotherapy and radiotherapy in clinical trials4. This effect is not due to p53 acting alone, as TA p73 is also induced by many chemotherapeutic agents and reinforces the apoptotic response to chemotherapy. Further investigations have revealed that mutant p53 associated with the Arg polymorphism is able to bind to and inactivate p7355 and block apoptosis. This means that the response to radiotherapy or chemotherapy may be diminished when p53 is mutated and codon 72 carries the Arg allele. This was confirmed in a clinical study involving patients with head and neck tumours receiving chemoradiation, where a more favourable response was seen in those retaining the wild-type Pro72 allele92. Taken together, the experimental and clinical studies suggest that this single nucleotide polymorphism is an important determinant of the response to chemotherapy, and that analysis of p53 genotype, both mutation and the polymorphism at codon 72, may provide a more reliable means of predicting response to these treatment modalities in the future. While p73 plays an important role in the response to many anticancer agents, the response can be blocked by mutant p53 molecules including 143A and 175H, with the 72 Arg form of these mutants inhibiting p73 more efficiently than the 72 Pro forms, again highlighting the importance of the codon 72 polymorphism in influencing outcome. Other genetic events upstream or downstream of p53 may also knockout the p53 pathways. This means that p53-dependent apoptosis may be active in only a small percentage of head and neck tumours. In addition, tumours that retain a functional

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p53 pathway may lose other basic apoptotic mechanisms as they progress towards continued proliferation and independence from intrinsic and extrinsic growth-control mechanisms. These include overexpression of genes that belong to the Bcl 2 family and there may be loss of the caspase-encoding genes. Tumours also frequently lose sensitivity to extrinsic apoptotic stimuli (such as Fas, tumour necrosis factor and TRAIL) as a means of escaping from the host immune response30 and apoptosis. As tumours harbour different defects in these pathways, and p53 also plays a role in cell-cycle arrest and repair as well as apoptosis, it is now clear that the response to radiotherapy or chemotherapy depends on the overall genetic background of the cell, rather than the presence or absence of a mutation affecting the p53 gene. The speed of growth of a tumour also affects the response to treatment, with rapidly proliferating tumours being more sensitive to these agents than slow-growing tumours. Another factor associated with radio-resistance is hypoxia, as radiation exerts part of its effects via the generation of free radicals. The key factors influencing the response to treatment are summarized in Fig. 7. As tumours eventually acquire defects in most of the key apoptotic pathways, they become increasingly resistant to radiotherapy and chemotherapy. 5-FU and cisplatin also exert some of their effects via p53-independent mechanisms, which is why they show some, albeit limited, efficacy against most head and neck tumours. This is also true for radiotherapy and explains why tumours with mutant p53 will respond, although the degree of response depends on the other pathways that remain functional within the cell. Tumours that respond poorly, or are resistant to radio- or chemotherapy, are presumed to have populations of cells that are very resistant to apoptosis. p53 and detection of residual cancer, field change and monitoring for recurrence

Surgical resection remains the principal treatment modality for the majority of patients presenting with advanced tumours and may also be used to treat early lesions. Successful surgery depends on excising a tumour with a good margin of normal tissue. As loco-regional recurrence remains the most common cause of treatment failure, and may occur even when the margins are considered to be clear after light microscopic examination,

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Fig. 7. The factors that influence the response to radiotherapy.

new diagnostics are required that can provide a more accurate assessment of the status of margins to ensure that appropriate adjuvant treatment can be given to those with molecular evidence of residual cancer. In order to devise more reliable ways of detecting the presence of residual malignant cells, a number of studies have used the p53 gene as a target for tumour cell detection, as this sequence is so frequently mutated in head and neck cancers. In essence, all approaches are based on finding the same signature p53 gene mutation in a tumour, and the surrounding normal or surgical margins that are pronounced tumour-free after examination by the pathologist. The first assay devised to detect residual cancer was very complex as it depended on the production of phage libraries containing p53 gene fragments isolated from the surgical margins of the tumour to be examined7. These gene fragments were amplified in bacteria and lawns containing holes or plaques (caused by lysis of the bacteria by the phage containing either mutant or wild-type p53), transferred to filters and probed with radiolabelled oligonucleotides specific for the signature p53 gene mutation present in the primary tumour, and then with a second oligonu-

cleotide probe specific for the wild-type gene (Fig. 8). This allows the percentage of residual tumour cells harbouring a p53 mutation to be estimated for mucosal and deep surgical margins. While this assay has exquisite sensitivity for tumour detection in the mucosal or deep margins, it may be 4–6 weeks before the test result is available to the clinician. This means that while this first-generation test has provided a valuable research tool7,74,95, simple, more rapid approaches are needed if these tests are to be used to influence the decision on what adjuvant treatment should be given to prevent tumour recurrence. To make progress in this area, new approaches have been identified that utilize gap-ligase chain technology33. For this method, two sets of primers that span the sequences on either side of the mutation are designed. One of the forward primers has a gap at the end next to the mutation. The key feature of this method is that when the nucleotide complementary to the p53 mutation (for example, T if the mutation is A) is provided in the reaction mixture, and the complementary primer is bound to the mutated sequence, an enzyme termed DNA ligase can add the missing nucleotide to the chain to fill the gap and ligate the primer sequences. Once

joined, this new strand serves as a template for ligation of a second set of complementary primers that can be labelled and detected using fluorescent dyes, with the increase in fluorescence measured quantitatively in real time. The ligation reaction is repeated over 30 cycles and serves to amplify the amount of mutant gene product (see Fig. 8 for an example). A preliminary report suggests that this method has a sensitivity for tumour cell detection that is close to that found with the phage plaque assay. These diagnostics are currently in clinical trial with the aim of establishing whether molecular detection of residual cancer can identify cases at high risk of loco-regional recurrence. If this association is confirmed, as radiotherapy does not always prevent recurrence when p53 mutation-positive residual carcinoma is present, these individuals can be offered the opportunity to participate in treatment trials where they receive other adjuvants in addition to radiotherapy, to minimize their risk of relapse. It is envisaged that anti-angiogenic agents that may help to prevent outgrowth of residual cancer cells, or small molecules that interfere with growth factor signalling would be ideal agents for forwarding to this type of study. Use of p53 mutations to identify precancerous fields

Molecular approaches can also be used to identify the presence of cells harbouring p53 mutations in tumour-distant, clinically normal oral mucosa to help identify those patients who develop cancers that are surrounded by large, potentially precancerous fields and are at risk of developing a second tumour75,76. In addition, it is now well recognized that a proportion of head and neck tumours develop in association with areas of erythropkia or leukoplakia. Many of these precursor lesions harbour the genetic aberrations associated with tumour development69 and there is good evidence that dysplastic lesions that harbour more of the aberrations associated with tumour development are more likely to progress to cancer75,84. The presence of a p53 gene mutation, or a deletion, may also provide information about the risk of transformation, as these aberrations are detected most frequently in severe dysplasia, and it is generally considered that high-grade dysplastic lesions have the greatest risk of progression to cancer22,98. The potential clinical applications based on knowledge of p53 status are summarized in Fig. 6.

The changing face of p53 in head and neck cancer

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or a new primary include concordant allelic imbalance at multiple chromosomal loci, the presence of shared microsatellite alleles, concordant p53 gene mutations and concordant X chromosome analysis (females only). A pilot study applying a combination of these markers has shown that concordant aberrations are frequently found in paired tumours. This suggests that many so-called second tumours are actually recurrences of an index lesion76. This highlights the need to develop new ways to eradicate the precancerous fields that frequently surround tumours to prevent the evolution of a second tumour if cure rates are to be improved. Targeting the p53 gene may provide one such therapeutic option for these precursor lesions. Therapeutic approaches involving modulation of p53 Gene therapy to replace/restore p53 function

Fig. 8. Methods for detection of residual cancer. The phage plaque assay identifies a signature mutation present in the tumour (A) and two surgical margins (B) and (C), but not for another case (D) with a distinct p53 mutation. The gap ligase assay also indicates the presence of residual tumour in a surgical margin (black line).

p53 and aneuploidy

p53 has often been considered to be associated with the development of aneuploidy (the presence of abnormal numbers of chromosomes). This is one of the most common defects associated with tumour development. Aneuploidy can be detected at very early stages of transformation and may be present in premalignant lesions although the number of abnormal chromosomes generally increases with tumour progression, and tumours with aggressive clinical behaviour are more likely to be aneuploid than their less malignant counterparts. Although it is clear that p53 inactivation can promote aneuploidy, there is increasing evidence that loss of p53 alone is not a primary cause of aneuploidy21 in that, while aneuploid tumours frequently harbour p53 gene mutations, loss of the Rb and p16 proteins may also facilitate aneu-

ploidy. Overexpression of the kinases associated with mitosis, for example the Aurora kinase, and centrosome duplication may also be associated with aneuploidy. Nevertheless, a defective p53 pathway may facilitate chromosomal instability by preventing arrest at the G1-S cell-cycle checkpoint defective apoptosis. Chromosome imbalances may also cause widespread changes in gene transcription, and these may give the developing tumour cells a selective advantage. Distinguishing whether a new lesion is a recurrence or a true second tumour

It is also possible to apply multiple molecular markers to shed light on the clonal origin of two tumours. Those that can be combined to help assess whether a second tumour is a recurrence of an index lesion

Several studies have attempted to restore the function of p53 by introducing the wild-type gene into tumour cells harbouring mutant p53. An alternative strategy aims to restore the function of mutant p53 protein and increase the amount of wildtype p53 available in the cell. As the p53 gene plays a key role in cellcycle regulation and induces apoptosis in tumour but not normal cells, restoring the function of this sequence by p53 gene transfer has been investigated as a means of eliminating tumour cells. Adenoviruses have been studied extensively in this respect, as these viruses enter cells via the widely distributed coxsackie and adenovirus receptors, which are used for primary attachment of the virus to the cell. These receptors are expressed at varying levels by head and neck squamous tumours. Preclinical studies using adenovirus carrying wild-type p53 (Adp53) showed significant inhibition of tumour growth and FREDERICK et al.27 demonstrated that replacement of the p53 gene using an adenoviral vector resulted in apoptosis, and that this was associated with a reduction in levels of c-myc and Bax, but no change in the levels of Bcl 2. Other studies have shown varying responses in apoptosis-related genes in head and neck tumour cell lines after p53 gene therapy99, reflecting the complicated genetic basis of this tumour type. Benefit has also been demonstrated in clinical studies involving patients with non-resectable tumours that were resistant to radio- or chemotherapy14,15,64. Several reports have demonstrated that injection of

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Adp53 directly into a tumour only gives rise to expression of wild-type p53 in the cells adjacent to the needle tract, due to the high interstitial tissue pressure in the tumour. This has been confirmed in studies evaluating the efficacy of this virus to eliminate residual tumour in deep tissues, the most common site for treatment failure67. Other problems associated with adenovirus-mediated approaches include toxic effects due to delivery of high dose78, and short-term expression of the transgene, most likely due to the cellular immune response to adenovirus. The hope was that it would be possible to combine the delivery of Adp53 with radiotherapy or chemotherapy, as wildtype p53 can induce apoptosis and is an important mediator of the effects of radiotherapy and chemotherapy. Re-introduction of the p53 gene into head and neck squamous cell carcinoma cells has been shown to restore radio- or chemosensitivity, but this is not always the case. In some studies restoration of the p53 gene increased radiosensitivity but not chemosensitivity and vice versa58. These studies have revealed that restoration of the function of the p53 gene often results in prolonged G1 or G2 arrest rather than apoptosis, due to increased expression of p21, Gadd45 and 14-3-3-s. This disappointing result is due to the fact that aberrations affecting many of the downstream effectors of apoptosis (Bax, Bak) and upstream regulators (hdm2, p14) also occur in these tumours such that apoptosis is frequently blocked. Subsequent studies investigated a conditionally replicating adenovirus, termed ONYX-015, designed to replicate only in cells that harbour mutated p53. Adenoviruses have two genes termed E1A and E1B that code for proteins that bind to and neutralize the Rb protein and p53, respectively. ONYX-015 has the E1B gene deleted, and it was originally considered that this virus would replicate selectively in tumour cells that lack the p53 pathway. Recent studies have demonstrated that it is loss of E1B-mediated export of viral RNA, rather than p53 inactivation, that prevents ONYX-015 replication in normal cells, with tumours that support the replication of ONYX-015 having abnormalities that allow export of viral RNA68. The ONYX-015 virus has been tested against many tumour types either as a single treatment, or in combination with chemotherapy or radiotherapy. To date, the most successful trial involving head and neck cancer patients was where intratumoural injection of ONYX-015 was given in combination with cisplatin and

5-FU. This resulted in a 63% overall response rate with 27% of cases demonstrating a full clinical response44. In this study, none of the responding tumours had progressed, whereas all non-injected tumours treated with chemotherapy alone progressed. ONYX-015 was also shown to be an efficient adjuvant treatment to radiotherapy with a greater activity in p53deficient tumours83. A subsequent clinical study delivering this virus by intratumoural injection for 40 head and neck cancer patients, who had recurrence/ relapse after prior conventional treatment, showed complete regression in 14% of cases, stable disease in 41% and progressive disease rate in 45%, indicating that the virus is a safe agent with modest antitumoural activity63. During another clinical study MORLEY et al.62 observed 15 oral squamous cell carcinoma patients with previously untreated squamous cell carcinoma in a phase II trial with treatment given via direct intra-tumoural injection before surgery. The tumour was then removed and evidence of viral replication in the tumour and surrounding normal tissue sought. This study established that ONYX-015 replicated in tumour but not in normal tissue, and that this agent has potential as a selective anticancer agent. As mutant p53 is also frequently associated with moderate or severe dysplastic lesions, ONYX-015 has also been used as a mouthwash in the treatment of oral dysplasia85 to good effect. Other clinical trials involving patients with ovarian and metastatic tumours have not shown objective clinical responses80,96 and further development of this agent has been abandoned. Approaches to silence mutant p53 and restore the function of p73

RNA interference (RNAi) is a powerful tool that can be used to silence genes by triggering the degradation of DNA. These siRNAs can be designed to exploit the difference between mutant and wild-type p53 sequences, to destroy mutant but not normal wild-type protein. MARTINEZ et al.56 demonstrated that siRNA can be used to suppress expression of p53 point mutations and restore chemosensitivity. This suggests that it may be possible to suppress the function of point-mutated p53 genes to provide a personalized anti-tumour therapy. Such an approach may be very useful for patients with Li-Fraumeni syndrome who carry one mutated p53 allele in the genome and have a very high risk of tumour development. Blocking mutant p53 may

restore DNA surveillance and prevent accumulation of the mutations that give rise to the cancers that develop in these patients. As there are p53 mutations (143H) that interact with p73 to inactivate p73, it may also be possible to use siRNA to knockout the function of these inhibitory p53 mutations and restore the pro-apoptotic function of p73. Use of p53 inhibitors to suppress the side effects of radiotherapy

KOMAROVA & GUDKOV46 hypothesized that as p53-mediated apoptosis is a major determinant of the side effects of cancer treatment, blocking expression of p53 should reduce the damage to normal tissues. This team isolated a chemical inhibitor of p53 termed pifithrin-a (PFT), an abbreviation for p-fifty-three inhibitor, and showed that this small molecule protected mouse fibroblasts from apoptosis induced by UV and gamma radiation, and different chemotherapeutic drugs. There are some concerns that even short-term suppression of p53 might result in the survival of genetically altered cells that otherwise would be eliminated by apoptosis. Approach to increase the activity of wildtype p53

p53 has long been considered a prime target for therapeutic modulation, and a number of methods have been devised that can turn on the p53 response without causing DNA damage. The most characterized p53 inhibitor is hdm2, which inhibits p53 activities through an autoregulatory feedback loop, such that blocking the p53–hdm2 interaction activates p53 and induces growth arrest and apoptosis. This constitutes an attractive strategy for activating p53, and two major approaches have been employed toward this goal: interference with the p53–hdm2 interaction and down-regulation of hdm2 expression. VASSILEV et al.97 reported that a class of small molecules, termed nutlins, that occupy the p53 binding pocket of hdm2, prevent this protein from binding to p53. They demonstrated that the activity of nutlins is greater in cancer cells that show increased expression of hdm2, and that these proteins can induce growth arrest and apoptosis. Other compounds that can restore the wild-type conformation of p53 have recently been isolated. For example, BYKOV et al.10 isolated a small molecule that selectively inhibits the growth of

The changing face of p53 in head and neck cancer tumour cells expressing mutant p53 by converting the mutant molecule back to a properly folded biological active form. This agent, termed p53 activation and induction of massive apoptosis (PRIMA1), was shown to inhibit the growth of tumour cells expressing a number of different p53 gene mutations, and to induce apoptosis and slow tumour growth in vivo. Other new approaches to enhance apoptosis

The majority of therapeutic agents exert their effects by inducing apoptosis, such that the genes and proteins that modulate this process are all considered to be ‘druggable’ targets. For example, iASPP is an inhibitory member of the ASPP family that blocks p53-dependent apoptosis and may contribute to resistance to the cytotoxic effects of cisplatin, radiation and UV, such that inhibiting the function of iASPP could provide an important new strategy for treating tumours that express wild-type p535. In conclusion, the era of molecular pathology is now on the horizon, and as p53 and its family members play a pivotal role in determining the response to treatment, in the future it seems likely that establishing the p53 mutation status of a tumour, the nature of the polymorphism at codon 72 and the levels of p73 and p63 will be required to predict this response. Molecular information can also be provided about the possibility of residual cancer after surgery and the existence of fields of damaged mucosa in which further foci of tumour may evolve. Understanding how the p53 family members interact also provides new opportunities to develop treatments that can restore these pathways to target and destroy cancer cells. Many agents are now entering clinical trials, and if their efficacy is confirmed this will provide an opportunity to devise new treatment schedules for head and neck cancer patients.

3.

4.

5.

6.

7.

8.

9.

References 1. Abbas T, White D, Hui L, Yoshida DA, Bargonetti J. Inhibition of human p53 basal transcription by down-regulation of protein kinase Cdelta. J Biol Chem 2004: 279: 9970–9977. 2. Ahomadegbe JC, Barrois M, Fogel S, Le Bihan ML, Douc-Rasy S, Duvillard P, Armand JP, Riou G. High incidence of p53 alterations (mutation, deletion, overexpression) in head and neck primary tumors and metastases; absence of correlation with clinical out-

10.

11.

come. Frequent protein overexpression in normal epithelium and in early noninvasive lesions. Oncogene 1995: 10: 1217–1227. Balz V, Scheckenbach K, Gotte K, Bockmuhl U, Petersen I, Bier H. Is the p53 inactivation frequency in squamous cell carcinomas of the head and neck underestimated? Analysis of p53 exons 2–11 and human papillomavirus 16/18 E6 transcripts in 123 unselected tumor specimens. Cancer Res 2003: 63: 1188–1191. Bergamaschi D, Gasco M, Hiller L, Sullivan A, Syed N, Trigante G, Yulug I, Merlano M, Numico G, Comino A, Attard M, Reelfs O, Gusterson B, Bell AK, Heath V, Tavassoli M, Farrell PJ, Smith P, Lu X, Crook T. p53 polymorphism influences response in cancer chemotherapy via modulation of p73-dependent apoptosis. Cancer Cell 2003: 3: 387–402. Bergamaschi D, Samuels Y, O’Neil NJ, Trigiante G, Crook T, Hsieh JK, O’Connor DJ, Zhong S, Campargue I, Tomlinson ML, Kuwabara PE, Lu X. iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human. Nat Genet 2003: 33: 162–167. Bradford CR, Zhu S, Ogawa H, Ogawa T, Ubell M, Narayan A, Johnson G, Wolf GT, Fisher SG, Carey TE. p53 mutation correlates with cisplatin sensitivity in head and neck squamous cell carcinoma lines. Head Neck 2003: 25: 654–661. Brennan JA, Mao L, Hruban RH, Boyle JO, Eby YJ, Koch WM, Goodman SN, Sidransky D. Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck. N Engl J Med 1995: 332: 429– 435. Bosch FX, Ritter D, Enders C, Flechtenmacher C, Abel U, Dietz A, Hergenhahn M, Weidauer H. Head and neck tumor sites differ in prevalence and spectrum of p53 alterations but these have limited prognostic value. Int J Cancer 2004: 111: 530– 538. Boyle JO, Hakim J, Koch W, van der Riet P, Hruban RH, Roa RA, Correo R, Eby YJ, Ruppert JM, Sidransky D. The incidence of p53 mutations increases with progression of head and neck cancer. Cancer Res 1993: 53: 4477–4480. Bykov VJ, Issaeva N, Shilov A, Hultcrantz M, Pugacheva E, Chumakov P, Bergman J, Wiman KG, Selivanova G. Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat Med 2002: 8: 282–288. Cabelguenne A, Blons H, de Waziers I, Carnot F, Houllier AM, Soussi T, Brasnu D, Beaune P,

12.

13.

14.

15.

16.

17.

18.

19.

20.

1135

Laccourreye O, Laurent-Puig P. p53 alterations predict tumor response to neoadjuvant chemotherapy in head and neck squamous cell carcinoma: a prospective series. J Clin Oncol 2000: 18: 1465–1473. Cai YC, Yang GY, Nie Y, Wang LD, Zhao X, Song YL, Seril DN, Liao J, Xing EP, Yang CS. Molecular alterations of p73 in human esophageal squamous cell carcinomas: loss of heterozygosity occurs frequently; loss of imprinting and elevation of p73 expression may be related to defective p53. Carcinogenesis 2000: 21: 683–689. Choi HR, Batsakis JG, Zhan F, Sturgis E, Luna MA, El-Naggar AK. Differential expression of p53 gene family members p63 and p7P3 in head and neck squamous tumorigenesis. Hum Pathol 2002: 33: 158–164. Clayman GL, El-Naggar AK, Lippman SM, Henderson YC, Frederick M, Merritt JA, Zumstein LA, Timmons TM, Liu TJ, Ginsberg L, Roth JA, Hong WK, Bruso P, Goepfert H. Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma. J Clin Oncol 1998: 16: 2221–2232. Clayman GL, Frank DK, Bruso PA, Goepfert H. Adenovirus-mediated wild-type p53 gene transfer as a surgical adjuvant in advanced head and neck cancers. Clin Cancer Res 1999: 5: 1715–1722. Cutilli T, Papola F, Di Emidio P, Leocata P, Corbacelli A. p53 mutations and chemoresistance in oromaxillofacial squamous-cell carcinomas. The results of a molecular genetics study of p53 in metastatic tumors and an evaluation of the response to neoadjuvant chemotherapeutic treatment. Minerva Stomatol 1998: 47: 1–9. Dai M, Clifford GM, le Calvez F, Castellsage X, Snijders PJ, Pawlita M, Herrero R, Hainaut P, Franceschi S. IARC Multicenter Oral Cancer Study Group.Human papillomavirus type 16 and TP53 mutation in oral cancer: matched analysis of the IARC multicenter study. Cancer Res 2004: 64: 468–471. Denissenko MF, Pao A, Tang M, Pfeifer GP. Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science 1996: 274: 430–432. D’Orazi G, Cecchinelli B, Bruno T, Manni I, Higashimoto Y, Saito S, Gostissa M, Coen S, Marchetti A, Del Sal G, Piaggio G, Fanciulli M, Appella E, Soddu S. Homeodomaininteracting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis. Nat Cell Biol 2002: 4: 11–19. Dumont PJ, Leu I-J, Pietra III ACD, George DL, Murphy M. The codon 72 polymorphic variants of p53 have mark-

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21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

Partridge et al.

edly different apoptotic potential. Nat Genet 2003: 33: 357–365. Duensing A, Duensing S. Guilt by association? p53 and the development of aneuploidy in cancer. Biochem Biophys Res Commun 2005: 331: 694–700. El-Naggar AK, Lai S, Luna MA, Zhou XD, Weber RS, Goepfert H, Batsakis JG. Sequential p53 mutation analysis of pre-invasive and invasive head and neck squamous carcinoma. Int J Cancer 1995: 64: 196–201. Eriksen JG, Buffa FM, Alsner J, Steiniche T, Bentzen SM, Overgaard J. Molecular profiles as predictive marker for the effect of overall treatment time of radiotherapy in supraglottic larynx squamous cell carcinomas. Radiother Oncol 2004: 72: 275– 282. Flores ER, Tsai KY, Crowley D, Sengupta S, Yang A, McKeon F, Jacks T. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 2002: 416: 560– 564. Fomenkov A, Huang YP, Topaloglu O, Brechman A, Osada M, Fomenkova T, Yuriditsky E, Trink B, Sidransky D, Ratovitski E. p63 alpha mutations lead to aberrant splicing of keratinocyte growth factor receptor in the Hay-Wells syndrome. J Biol Chem 2003: 278: 23906–23914. Fouret P, Temam S, Charlotte F, Lacau-St-Guily J. Tumour stage, node stage, p53 gene status, and bcl-2 protein expression as predictors of tumour response to platin-fluorouracil chemotherapy in patients with squamouscell carcinoma of the head and neck. Br J Cancer 2002: 87: 1390–1395. Frederick MJ, Holton PR, Hudson M, Wang M, Clayman GL. Expression of apoptosis-related genes in human head and neck squamous cell carcinomas undergoing p53-mediated programmed cell death. Clin Cancer Res 1999: 5: 361–369. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, Zahurak ML, Daniel RW, Viglione M, Symer DE, Shah KV, Sidransky D. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Natl Cancer Inst 2000: 92: 709–720. Greenblatt MS, Bennett WP, Hollstein M, Haris CC. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 1994: 54: 4855–4878. Gudkov AV, Komarova EA. The role of p53 in determining sensitivity to radiotherapy. Nat Rev Cancer 2003: 3: 117–129. Hafkamp HC, Speel EJ, Haesevoets A, Bot FJ, Dinjens WN, Ramaekers FC, Hopman AH, Manni JJ. A subset of head and neck squamous cell carcino-

32.

33.

34.

35.

36.

37.

38.

39. 40.

41.

42. 43.

mas exhibits integration of HPV 16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8. Int J Cancer 2003: 107: 394–400. Hall PA, Lane DP. p53 in tumour pathology: can we trust immunohistochemistry? – Revisited! J Pathol 1994: 172: 1–4. Harden SV, Thomas DC, Benoit N, Minhas K, Westra WH, Califano JA, Koch W, Sidransky D. Real-time gap ligase chain reaction: a rapid semi-quantitative assay for detecting p53 mutation at low levels in surgical margins and lymph nodes from resected lung and head and neck tumors. Clin Cancer Res 2004: 10: 2379–2385. Herrero R. Chapter 7: Human papillomavirus and cancer of the upper aerodigestive tract. J Natl Cancer Inst Monogr 2003: 31: 47–51. Hibi K, Trink B, Patturajan M, Westra WH, Caballero OL, Hill DE, Ratovitski EA, Jen J, Sidransky D. AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci USA 2000: 97: 5462–5467. Honeycutt KA, Koster MI, Roop DR. Genes involved in stem cell fate decisions and commitment to differentiation play a role in skin disease. J Investig Dermatol Symp Proc 2004: 9: 261–268. Hong Y, Miao X, Zhang X, Ding F, Luo A, Guo Y, Tan W, Liu Z, Lin D. The role of p53 and MDM2 polymorphisms in the risk of esophageal squamous cell carcinoma. Cancer Res 2005: 65: 9582–9587. Hu H, Xia SH, Li AD, Xu X, Cai Y, Han YL, Wei F, Chen BS, Huang XP, Han YS, Zhang JW, Zhang X, Wu M, Wang MR. Elevated expression of p63 protein in human esophageal squamous cell carcinomas. Int J Cancer 2002: 102: 580–583. Jamieson ER, Lippard SJ. Structure and processing of cisplatin-DNA adducts. Chem Rev 1999: 98: 2467–2498. Jeffrey PD, Gorina S, Pavletich NP. Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science 1995: 267: 1498–1502. Jost CA, Marin MC, Kaelin Jr WG. p73 is a simian [correction of human] p53-related protein that can induce apoptosis. Nature 1997: 389: 191–194. Kaelin WG. The p53 gene family. Oncogene 1999: 18: 7701–7715. Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Lelias JM, Dumont X, Ferrara P, McKeon F, Caput D. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 1997: 90: 809–819.

44. Khuri FR, Nemunaitis J, Ganly I, Arseneau J, Tannock IF, Romel L, Gore M, Ironside J, MacDougall RH, Heise C, Randlev B, Gillenwater AM, Bruso P, Kaye SB, Hong WK, Kirn DH. A controlled trial of intratumoural ONYX-015 a selectively-replicating adenovirus in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 2000: 6: 879–885. 45. Koch WM, Lango M, Sewell D, Zahurak M, Sidransky D. Head and neck cancer in nonsmokers: a distinct clinical and molecular entity. Laryngoscope 1999: 109: 1544–1551. 46. Komarova EA, Gudkov AV. Chemoprotection from p53-dependent apoptosis: potential clinical applications of the p53 inhibitors. Biochem Pharmacol 2001: 62: 657–667. 47. Koster M, Huntzinger KA, Roop DR. Epidermal differentiation: transgenic/knockout mouse models reveal genes involved in stem cell fate decisions and commitment to differentiation. J Invest Dermatol Symp Proc 2002: 7: 41–45. 48. Kress S, Sutter C, Strickland PT, Mukhtar H, Schweizer J, Schwarz M. Carcinogen-specific mutational pattern in the p53 gene in ultraviolet B radiation-induced squamous cell carcinomas of mouse skin. Cancer Res 1992: 52: 6400–6413. 49. Kropveld A, Rozemuller EH, Leppers FG, Scheidel KC, de Weger RA, Koole R, Hordijk GJ, Slootweg PJ, Tilanus MG. Sequencing analysis of RNA and DNA of exons 1 through 11 shows p53 gene alterations to be present in almost 100% of head and neck squamous cell cancers. Lab Invest 1999: 79: 347–353. 50. Lane DP. Cancer. p53, guardian of the genome. Nature 1992: 358: 15–16. 51. Larsen AK, Escargueil AE, Skladanowski A. From DNA damage to G2 arrest: the many roles of topoisomerase II. Prog Cell Cycle Res 2003: 5: 295– 300. 52. Lee SK, Kim SB, Kim JS, Moon CH, Han MS, Lee BJ, Chung DK, Min YJ, Park JH, Choi DH, Cho HR, Park SK, Park JW. Butyrate response factor 1 enhances cisplatin sensitivity in human head and neck squamous cell carcinoma cell lines. Int J Cancer 2005: 117: 32–40. 53. Leng K, Schlien S, Bosch FX. Refined characterization of head and neck squamous cell carcinomas expressing a seemingly wild-type p53 protein. J Oral Pathol Med 2006: 35: 19–24. 54. Mantovani F, Banks L. The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene 2001: 20: 7874–7887. 55. Marin MC, Jost CA, Brooks LA, Irwin MS, O’Nions J, Tidy JA, James

The changing face of p53 in head and neck cancer

56.

57.

58.

59.

60. 61. 62.

63.

64.

65.

N, McGregor JM, Harwood CA, Yulug IG, Vousden KH, Allday MJ, Gusterson B, Ikawa S, Hinds PW, Crook T, Kaelin WG. A common polymorphism acts as an intragenic modifier of mutant p53 behaviour. Nat Genet 2000: 25: 47–54. Martinez LA, Naguibneva I, Lehrmann H, Vervisch A, Tche´nio T, Lozano G, Harel-Bellan A. Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways. Proc Natl Acad Sci USA 2002: 99: 14849–14854. McCluggage WG, Connolly LE, McGregor G, Hyland PL, Hall PA. A strategy for defining biologically relevant levels of p53 protein expression in clinical samples with reference to endometrial neoplasia. Int J Gynecol Pathol 2005: 24: 307–312. Medema RH, Klompmaker R, Smits VA, Rijksen G. p21waf1 can block cells at two points in the cell cycle, but does not interfere with processive DNA-replication or stress-activated kinases. Oncogene 1998: 16: 431–441. Melino G, Bernassola F, Ranalli M, Yee K, Zong WX, Corazzari M, Knight RA, Green DR, Thompson C, Vousden KH. p73 induces apoptosis via PUMA transactivation and Bax mitochondrial translocation. J Biol Chem 2004: 279: 8076–8083. Melino G. p73, the ‘assistant’ guardian of the genome? Ann N Y Acad Sci 2003: 1010: 9–15. Moll UM, Slade N. p63 and p73: roles in development and tumor formation. Mol Cancer Res 2004: 2: 371–386. Morley S, MacDonald G, Kirn D, Kaye S, Brown R, Soutar D. The dl1520 virus is found preferentially in tumor tissue after direct intratumoral injection in oral carcinoma. Clin Cancer Res 2004: 10: 4357–4362. Nemunaitis J, Khuri F, Ganly I, Arseneau J, Posner M, Vokes E, Kuhn J, McCarty T, Landers S, Blackburn A, Romel L, Randlev B, Kaye S, Kirn D. Phase II trial of intratumoral administration of ONYX015, a replication-selective adenovirus, in patients with refractory head and neck cancer. J Clin Oncol 2001: 19: 289–298. Nemunaitis J, Swisher SG, Timmons T, Connors D, Mack M, Doerksen L, Weill D, Wait J, Lawrence DD, Kemp BL, Fossella F, Glisson BS, Hong WK, Khuri FR, Kurie JM, Lee JJ, Lee JS, Nguyen DM, Nesbitt JC, Perez-Soler R, Pisters KM, Putnam JB, Richli WR, Shin DM, Walsh GL, Merritt J, Roth J. Adenovirus mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. J Clin Oncol 2000: 18: 609–622. Nenutil R, Smardova J, Pavlova S, Hanzelkova Z, Muller P, Fabian P,

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

Hrstka R, Janotova P, Radina M, Lane DP, Coates PJ, Vojtesek B. Discriminating functional and non-functional p53 in human tumours by immunohistochemistry. J Pathol 2005: 207: 251–259. Nylander K, Nilsson P, Mehle C, Roos G. p53 mutations, protein expression and cell proliferation in squamous cell carcinomas of the head and neck. Br J Cancer 1995: 71: 826–830. Oakley R, Phillips E, Hooper R, Wilson D, Partridge M. A preclinical model of minimal residual cancer in the muscle highlights challenges associated with adenovirus-mediated p53 gene transfer. Clin Cancer Res 2002: 8: 1984–1994. O’Shea CC, Johnson L, Bagus B, Choi S, Nicholas C, Shen A, Boyle L, Pandey K, Soria C, Kunich J, Shen Y, Habets G, Ginzinger D, McCormick F. Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumour selectivity. Cancer Cell 2004: 6: 611–623. Partridge M, Emilion G, Pateromichelakis S, Phillips E, Langdon J. Field cancerisation of the oral cavity: comparison of the spectrum of molecular alterations in cases presenting with both dysplastic and malignant lesions. Eur J Cancer Oral Oncol 1997: 33: 332– 337. Partridge M, Emilion G, Pateromichelakis S, A’Hern R, Phillips E, Langdon J. Allelic imbalance at chromosomal loci implicated in the pathogenesis of oral precancer, cumulative loss and its relationship with progression to cancer. Eur J Cancer Oral Oncol 1998: 34: 77–83. Partridge M, Emilion G, Pateromichelakis S, A’Hern R, Lee G, Phillips E, Langdon J. The prognostic significance of allelic imbalance at key chromosomal loci in oral cancer. Br J Cancer 1999: 79: 1821–1827. Partridge M, Emilion G, Falworth M, A’Hern R, Phillips E, Pateromichelakis S, Langdon J. Patient-specific mutation databases for oral cancer. Int J Cancer 1999: 84: 284–292. Partridge M, Kiguwa S, Emilion G, Pateromichelakis S, A’Hern R, Langdon JD. New insights into p53 protein stabilisation in oral squamous cell carcinoma. Eur J Cancer 1999: 35: 45–55. Partridge M, Li S-R, Pateromichelakis S, Francis RA, Phillips E, Huang X-H, Tesfa-Selase F, Langdon JD. Detection of minimal residual cancer to investigate why oral tumours recur despite seemingly adequate treatment. Clin Cancer Res 2000: 6: 2718–2725. Partridge M, Pateromichelakis S, Phillips E, Emilion GG, A’Hern RP, Langdon JD. A case-control study confirms that microsatellite assay can

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

1137

identify patients at risk of developing oral SCC within a field of cancerisation. Cancer Res 2000: 60: 3893–3898. Pateromichelakis S, Farahini M, Phillips E, Partridge M. Molecular analysis of paired tumours: Time to start treating the field. Oral Oncol 2005: 41: 916–926. Partridge M, Gaballah K, Huang X. Molecular markers for diagnosis and prognosis. Cancer Metast Rev 2005: 24: 71–85. Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg A, Gao GP, Wilson JM, Batshaw ML. Fatal systemic inflammatory response in an ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 2003: 80: 148–158. Ratovitski EA, Patturajan M, Hibi K, Trink B, Yamaguchi K, Sidransky D. p53 associates with and targets Np63 into a protein degradation pathway. Proc Natl Acad Sci USA 2001: 98: 1817– 1822. Reid TR, Freeman S, Post L, McCormick F, Sze DY. Effects of ONYX-015 among metastatic colorectal cancer patients that have failed prior treatment with 5-FU/leucovorin. Cancer Gene Ther 2005: 12: 673–681. Rozemuller EH, Kropveld A, Kreyveld E, Leppers FG, Scheidel K. Sensitive detection of p53 mutation: analysis by direct sequencing and multisequence analysis. Cancer Detect Prev 2001: 25: 109–116. Rocco JW, Leong C-O, Kuperwasser N, DeYoung MP, Ellison LW. p63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 2006: 9: 45–56. Rogulski KR, Freytag SO, Zhang K, Gilbert JD, Paielli DL, Kim JH, Heise CC, Kirn DH. In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy. Cancer Res 2000: 60: 1193– 1196. Rosin MP, Cheng X, Poh CF, Lam WL, Huang Y, Lovas J, Berean K, Epstein JB, Priddy R, Le ND, Zhang L. Use of allelic loss to predict malignant risk for low-grade oral epithelial dysplasia. Clin Cancer Res 2000: 6: 357–362. Rudin CM, Cohen EEW, Papadimitrakopoulou VA. An attenuated adenovirus ONYX-015 as mouthwash therapy for premalignant oral dysplasia. J Clin Oncol 2003: 21: 4546–4552. Samuels-Lev Y, O’Connor DJ, Bergamaschi D, Trigiante G, Hsieh JK, Zhong S, Campargue I, Naumovski. Crook T, Lu X. ASPP proteins specifically stimulate the apoptotic function of p53. Mol Cell 2001: 8: 781–794. Schmale H, Bamberger C. A novel protein with strong homology to the

1138

88.

89.

90.

91.

92.

93.

Partridge et al.

tumor suppressor p53. Oncogene 1997: 15: 1363–1367. Sheikh MS, Burns TF, Huang Y, Wu GS, Amundson S, Brooks KS, Fornace AJ, el-Deiry WS. p53-dependent and -independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor alpha. Cancer Res 1998: 58: 1593–1598. Shin HJ, Kalapurakal SK, Lee JJ, Ro JY, Hong WK, Lee JS. Comparison of p53 immunoreactivity in fresh-cut versus stored slides with and without microwave heating. Mod Pathol 1997: 10: 224–230. Sniezek JC, Matheny KE, Westfall MD, Pietenpol JA. Dominant Negative p63 Isoform Expression in Head and Neck Squamous Cell Carcinoma. Laryngoscope 2004: 114: 2063–2072. Soussi T, Asselain B, Hamroun D, Kato S, Ishioka C, Claustres M, Beroud C. Meta-analysis of the p53 mutation database for mutant p53 biological activity reveals a methodologic bias in mutation detection. Clin Cancer Res 2006: 12: 62–69. Sullivan A, Syed N, Gasco M, Bergamaschi D, Trigiante G, Attard M, Hiller L, Farrell PJ, Smith P, Lu X, Crook T. Polymorphism in wild-type p53 modulates response to chemotherapy in vitro and in vivo. Oncogene 2004: 23: 3328–3337. Townsend PA, Scarabelli TM, Davidson SM, Knight RA, Latchman DS, Stephanou A. STAT-1 interacts with p53 to enhance DNA damageinduced apoptosis. J Biol Chem 2004: 279: 5811–5820.

94. van Houten VM, Leemans CR, Kummer JA, Dijkstra J, Kuik DJ, van den Brekel MW, Snow GB, Brakenhoff RH. Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: a prospective study. Clin Cancer Res 2004: 10: 3614– 3620. 95. van Houten VM, Snijders PJ, van den Brekel MW, Kummer JA, Meijer CJ, van Leeuwen B, Denkers F, Smeele LE, Snow GB, Brakenhoff RH. Biological evidence that human papillomaviruses are etiologically involved in a subgroup of head and neck squamous cell carcinomas. Int J Cancer 2001: 93: 232–235. 96. Vasey PA, Shulman LN, Campos S, Davis J, Gore M, Johnston S, Kirn DH, O’Neill V, Siddiqui N, Seiden MV, Kaye SB. Phase I trial of intraperitoneal injection of the E1B-55-kDgene-deleted adenovirus ONYX-015 (dl1520) given on days 1 through 5 every 3 weeks in patients with recurrent/refractory epithelial ovarian cancer. J Clin Oncol 2002: 20: 1562– 1569. 97. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA. In vivo activation of the p53 pathway by smallmolecule antagonists of MDM2. Science 2004: 303: 844–848. 98. Waridel F, Estreicher A, Bron L, Flaman J-M, Fontolliet C, Monnier P, Frebourg F, Iggo R. Field cancerisation and polyclonal p53 mutation in the upper aero-digestive tract. Oncogene 1997: 14: 163–169.

99. Wen WH, Bernstein L, Lescallett J, Beazer-Barclay Y, Sullivan-Halley J, White M. Comparison of TP53 mutations identified by oligonucleotide microarray and conventional DNA sequence analysis. Cancer Res 2000: 60: 2716–2722. 100. Wiest T, Schwarz E, Enders C, Flechtenmacher C, Bosch FX. Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control. Oncogene 2002: 21: 1510–1517. 101. Yamazaki Y, Chiba I, Hirai A, Sugiura C, Notani K, Kashiwazaki H, Tei K, Totsuka Y, Fukuda H. Specific p53 mutations predict poor prognosis in oral squamous cell carcinoma. Oral Oncol 2003: 39: 163–169. 102. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D, McKeon F. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998: 2: 305–316. 103. Zhu JJ, Jiang J, Zhou W, Chen X. The potential tumor suppressor p73 differentially regulates cellular p53 target genes. Cancer Res 1998: 58: 5061–5065. Address: Max Partridge Kings College Hospital Maxillofacial Unit Kings College London Denmark Hill London SE5 8RX UK E-mail: [email protected]