The p53 tumour suppressor gene

The cell cycle is composed of a series of steps which can be negatively or positively regulated by various factors. Chief among the negative regulators is the p53 protein. Alteration or inactivation of p53 by mutation, or by its interactions with oncogene products of DNA tumour viruses, can lead to cancer. These mutations seem to be the most common genetic change in human cancers.     

NUMB controls p53 tumour suppressor activity

NUMB is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family. NUMB is also an endocytic protein, and the NOTCH-NUMB counteraction has been linked to this function. There might be, however, additional functions of NUMB, as witnessed by its proposed role as a tumour suppressor in breast cancer. Here we describe a previously unknown function for human NUMB as a regulator of tumour protein p53 (also known as TP53). NUMB enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. This results in increased p53 protein levels and activity, and in regulation of p53-dependent phenotypes. In breast cancers there is frequent loss of NUMB expression. We show that, in primary breast tumour cells, this event causes decreased p53 levels and increased chemoresistance. In breast cancers, loss of NUMB expression causes increased activity of the receptor NOTCH. Thus, in these cancers, a single event-loss of NUMB expression-determines activation of an oncogene (NOTCH) and attenuation of the p53 tumour suppressor pathway. Biologically, this results in an aggressive tumour phenotype, as witnessed by findings that NUMB-defective breast tumours display poor prognosis. Our results uncover a previously unknown tumour suppressor circuitry.     

Localization of gene for human p53 tumour antigen to band 17p13

Recently the gene for the cellular tumour antigen p53, a phosphoprotein found in increased concentration in a variety of human cells, had been mapped to region 17q22 by in situ hybridization techniques and has been shown to translocate to the chromosome carrying the translocation [t(15; 17)] associated with acute promyelocytic leukaemia (APL). Based on this finding it has been postulated that this gene has a role in the pathogenesis of APL. Here we present evidence that the gene for p53 is not located on the long arm of chromosome 17, but maps to band 17p13. We therefore suggest that this gene is not directly involved in the chromosome translocation observed in APL.     

Selective activation of p53-mediated tumour suppression in high-grade tumours

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related death worldwide, with an overall 5-year survival rate of only 10-15%. Deregulation of the Ras pathway is a frequent hallmark of NSCLC, often through mutations that directly activate Kras. p53 is also frequently inactivated in NSCLC and, because oncogenic Ras can be a potent trigger of p53 (ref. 3), it seems likely that oncogenic Ras signalling has a major and persistent role in driving the selection against p53. Hence, pharmacological restoration of p53 is an appealing therapeutic strategy for treating this disease. Here we model the probable therapeutic impact of p53 restoration in a spontaneously evolving mouse model of NSCLC initiated by sporadic oncogenic activation of endogenous Kras. Surprisingly, p53 restoration failed to induce significant regression of established tumours, although it did result in a significant decrease in the relative proportion of high-grade tumours. This is due to selective activation of p53 only in the more aggressive tumour cells within each tumour. Such selective activation of p53 correlates with marked upregulation in Ras signal intensity and induction of the oncogenic signalling sensor p19(ARF)( )(ref. 6). Our data indicate that p53-mediated tumour suppression is triggered only when oncogenic Ras signal flux exceeds a critical threshold. Importantly, the failure of low-level oncogenic Kras to engage p53 reveals inherent limits in the capacity of p53 to restrain early tumour evolution and in the efficacy of therapeutic p53 restoration to eradicate cancers.     

The pathological response to DNA damage does not contribute to p53-mediated tumour suppression

The p53 protein has a highly evolutionarily conserved role in metazoans as 'guardian of the genome', mediating cell-cycle arrest and apoptosis in response to genotoxic injury. In large, long-lived animals with substantial somatic regenerative capacity, such as vertebrates, p53 is an important tumour suppressor--an attribute thought to stem directly from its induction of death or arrest in mutant cells with damaged or unstable genomes. Chemotherapy and radiation exposure both induce widespread p53-dependent DNA damage. This triggers potentially lethal pathologies that are generally deemed an unfortunate but unavoidable consequence of the role p53 has in tumour suppression. Here we show, using a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, that the p53-mediated pathological response to whole-body irradiation, a prototypical genotoxic carcinogen, is irrelevant for suppression of radiation-induced lymphoma. In contrast, delaying the restoration of p53 function until the acute radiation response has subsided abrogates all of the radiation-induced pathology yet preserves much of the protection from lymphoma. Such protection is absolutely dependent on p19(ARF)--a tumour suppressor induced not by DNA damage, but by oncogenic disruption of the cell cycle.     

Mutations in the p53 gene occur in diverse human tumour types

The p53 gene has been a constant source of fascination since its discovery nearly a decade ago. Originally considered to be an oncogene, several convergent lines of research have indicated that the wild-type gene product actually functions as a tumour suppressor gene. For example, expression of the neoplastic phenotype is inhibited, rather than promoted, when rat cells are transfected with the murine wild-type p53 gene together with mutant p53 genes and/or other oncogenes. Moreover, in human tumours, the short arm of chromosome 17 is often deleted. In colorectal cancers, the smallest common region of deletion is centred at 17p13.1; this region harbours the p53 gene, and in two tumours examined in detail, the remaining (non-deleted) p53 alleles were found to contain mutations. This result was provocative because allelic deletion coupled with mutation of the remaining allele is a theoretical hallmark of tumour-suppressor genes. In the present report, we have attempted to determine the generality of this observation; that is, whether tumours with allelic deletions of chromosome 17p contain mutant p53 genes in the allele that is retained. Our results suggest that (1) most tumours with such allelic deletions contain p53 point mutations resulting in amino-acid substitutions, (2) such mutations are not confined to tumours with allelic deletion, but also occur in at least some tumours that have retained both parental 17p alleles, and (3) p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene. These results suggest that p53 mutations play a role in the development of many common human malignancies.     

Participation of p53 cellular tumour antigen in transformation of normal embryonic cells

The cellular tumour antigen p53 is found at elevated levels in a wide variety of transformed cells (for reviews see refs 1, 2). Very little is yet known about the precise relationship of p53 to malignant transformation. Although the increase in p53 levels could be a secondary by-product of the transformed state, it is equally possible that p53 is actively involved in altering cellular growth properties, especially as it has been implicated in the regulation of normal cell proliferation. We sought to test whether p53 could behave in a manner similar to known genes in a biological test system, and we demonstrate here that p53 can cooperate with the activated Ha-ras oncogene to transform normal embryonic cells. The resultant foci contain cells of a markedly altered morphology which produce high levels of p53. Cell lines established from such foci elicit tumours in syngeneic animals.     

Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants

The c-Myc oncoprotein promotes proliferation and apoptosis, such that mutations that disable apoptotic programmes often cooperate with MYC during tumorigenesis. Here we report that two common mutant MYC alleles derived from human Burkitt's lymphoma uncouple proliferation from apoptosis and, as a result, are more effective than wild-type MYC at promoting B cell lymphomagenesis in mice. Mutant MYC proteins retain their ability to stimulate proliferation and activate p53, but are defective at promoting apoptosis due to a failure to induce the BH3-only protein Bim (a member of the B cell lymphoma 2 (Bcl2) family) and effectively inhibit Bcl2. Disruption of apoptosis through enforced expression of Bcl2, or loss of either Bim or p53 function, enables wild-type MYC to produce lymphomas as efficiently as mutant MYC. These data show how parallel apoptotic pathways act together to suppress MYC-induced transformation, and how mutant MYC proteins, by selectively disabling a p53-independent pathway, enable tumour cells to evade p53 action during lymphomagenesis.     

Cooperation between gene encoding p53 tumour antigen and ras in cellular transformation

The protein p53 is highly expressed in a large variety of transformed cell types originating from diverse species. These include cells transformed by Simian virus 40 (SV40), adenovirus and Abelson virus, as well as a variety of chemically transformed cells. Substantial amounts of p53 are also present in certain non-transformed cells, for example, some embryonic tissues. The protein may be localized in different cellular compartments in normal and transformed cells. The strong correlation between tumorigenicity and high levels of p53 suggests an important role of p53 in tumorigenesis. We report here experiments in which we have co-transfected the murine cellular gene encoding for p53 with a ras gene into primary rat embryo fibroblasts. Our results indicate that the p53-encoding gene can play a causal role in the conversion of normal fibroblasts into tumorigenic cells.     

Growth regulation of a cellular tumour antigen, p53, in nontransformed cells

Many transformed cells in culture have been found to express elevated levels of a cellular tumour antigen, termed p53. This protein has also been implicated in the regulation of cellular growth. For these reasons experiments were designed to examine the expression of p53 as quiescent cultures of nontransformed 3T3 fibroblasts were stimulated to reenter the cell cycle. Synchronous populations of cells were obtained by releasing a culture from density-dependent inhibition of growth with the addition of fresh serum. Steady-state levels of p53 protein and mRNA were measured as a function of time after addition of serum to quiescent cultures and the rate of synthesis of p53 protein was analysed at a number of time points. The results, reported here, demonstrate an increase in the synthesis and steady-state levels of p53 protein and mRNA prior to DNA synthesis in late G1, and suggest a role for p53 in the progression of cells from a growth-arrested state to an actively dividing state.     

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.     

Clonal expansion of p53 mutant cells is associated with brain tumour progression.

Tumour progression is a fundamental feature of the biology of cancer. Cancers do not arise de novo in their final form, but begin as small, indolent growths, which gradually acquire characteristics associated with malignancy. In the brain, for example, low-grade tumours (astrocytomas) evolve into faster growing, more dysplastic and invasive high-grade tumours (glioblastomas). To define the genetic events underlying brain tumour progression, we analysed the p53 gene in ten primary brain tumour pairs. Seven pairs consisted of tumours that were high grade both at presentation and recurrence (group A) and three pairs consisted of low-grade tumours that had progressed to higher grade tumours (group B). In group A pairs, four of the recurrent tumours contained a p53 gene mutation; in three of them, the same mutation was found in the primary tumour. In group B pairs, progression to high grade was associated with a p53 gene mutation. A subpopulation of cells were present in the low-grade tumours that contained the same p53 gene mutation predominant in the cells of the recurrent tumours that had progressed to glioblastoma. Thus, the histological progression of brain tumours was associated with a clonal expansion of cells that had previously acquired a mutation in the p53 gene, endowing them with a selective growth advantage. These experimental observations strongly support Nowell's clonal evolution model of tumour progression.     

Specific interaction between the p53 cellular tumour antigen and major heat shock proteins

The protein p53 is capable of participating in neoplastic transformation and can form specific complexes with the large-T antigen of simian virus 40 (SV40). This interaction probably results in the stabilization of p53 (refs 7,8) and may contribute to SV40-mediated transformation. Several non-SV40-transformed cells also exhibit a stabilized p53 which is present in elevated levels. Recently, this stabilization was shown to coincide with the ability to precipitate a polypeptide (p68) of relative molecular mass (Mr) 68,000-70,000 by anti-p53 monoclonal antibodies. We now report that this co-precipitation indeed represents a specific complex between the two proteins; the complex sediments on a sucrose gradient as a relatively broad peak of 10-14S and can be dissociated in vitro. Furthermore, p68 is the HSP70 heat shock protein cognate, found in elevated levels in a p53-overproducing cell line. On heat-shock treatment of such overproducers, p53 also forms a complex with the related highly inducible HSP68.     

Antibiotic-mediated antagonism leads to a bacterial game of rock-paper-scissors in vivo

Colicins are narrow-spectrum antibiotics produced by and active against Escherichia coli and its close relatives. Colicin-producing strains cannot coexist with sensitive or resistant strains in a well-mixed culture, yet all three phenotypes are recovered in natural populations. Recent in vitro results conclude that strain diversity can be promoted by colicin production in a spatially structured, non-transitive interaction, as in the classic non-transitive model rock-paper-scissors (RPS). In the colicin version of the RPS model, strains that produce colicins (C) kill sensitive (S) strains, which outcompete resistant (R) strains, which outcompete C strains. Pairwise in vitro competitions between these three strains are resolved in a predictable order (C beats S, S beats R, and R beats C), but the complete system of three strains presents the opportunity for dynamic equilibrium. Here we provide conclusive evidence of an in vivo antagonistic role for colicins and show that colicins (and potentially other bacteriocins) may promote, rather than eliminate, microbial diversity in the environment.     

Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene

The FBXW7/hCDC4 gene encodes a ubiquitin ligase implicated in the control of chromosome stability. Here we identify the mouse Fbxw7 gene as a p53-dependent tumour suppressor gene by using a mammalian genetic screen for p53-dependent genes involved in tumorigenesis. Radiation-induced lymphomas from p53+/- mice, but not those from p53-/- mice, show frequent loss of heterozygosity and a 10% mutation rate of the Fbxw7 gene. Fbxw7+/- mice have greater susceptibility to radiation-induced tumorigenesis, but most tumours retain and express the wild-type allele, indicating that Fbxw7 is a haploinsufficient tumour suppressor gene. Loss of Fbxw7 alters the spectrum of tumours that develop in p53 deficient mice to include a range of tumours in epithelial tissues such as the lung, liver and ovary. Mouse embryo fibroblasts from Fbxw7-deficient mice, or wild-type mouse cells expressing Fbxw7 small interfering RNA, have higher levels of Aurora-A kinase, c-Jun and Notch4, but not of cyclin E. We propose that p53-dependent loss of Fbxw7 leads to genetic instability by mechanisms that might involve the activation of Aurora-A, providing a rationale for the early occurrence of these mutations in human cancers.