HUMAN MUTATION 21:321^326 (2003)

p53 REVIEW ARTICLE

Disrupting TP53 in Mouse Models of Human Cancers John M. Parant and Guillermina Lozanon Department of Molecular Genetics, Section of Cancer Genetics, University of Texas M.D. Anderson Cancer Center, Houston, Texas For the p53 Special Issue Manipulation of the mouse genome allows emulation of the genetic defects that give rise to human cancers and evaluation of the cooperating nature of different mutations in the transformation of distinct cell types. Here we review the generation of mice with specific missense mutations in p53 (TP53) and disruption of the p53 pathway by deletion of p53 inhibitors. Missense mutations in the DNA binding domain result in viable mice with gain-of-function and dominant negative phenotypes. Loss of either of the p53 inhibitors mdm2 or mdm4 gives rise to a p53-dependent embryonic lethal phenotype. A cell can thus tolerate the absence of p53 function but not excess p53 function, a characteristic that is being exploited in the treatment of human cancers. Hum Mutat 21:321–326, 2003. r 2003 Wiley-Liss, Inc. KEY WORDS:

p53; TP53; cancer; tumor; mouse; animal model; MDM2; MDM4; suppressor; oncogene

DATABASES:

TP53 – OMIM: 191170; GenBank: NM_000546 (mRNA) http://p53.curie.fr/ (p53 Web Site at Institut Curie) www.iarc.fr/p53 (IARC p53 Mutation Database) MDM2 – OMIM: 164785 MDM4 – OMIM: 602704

INTRODUCTION

The ability to delete specific genes or to alter a single nucleotide in a specific gene has allowed biologists to manipulate the mouse genome and thus develop more accurate models of cancer. This review will begin by discussing a few examples of mouse models that have provided great insight into our understanding of human cancer, but will focus on the importance of missense mutations on p53 (TP53; MIM# 191170) function in tumorigenesis and on the role of two critical p53 inhibitors, MDM2 (MIM# 164785) and MDM4 (MIM# 602704). MAINTAINING TUMOR CELL PROLIFERATION

Mice that overexpress oncogenes or with loss or mutation of tumor suppressor genes have given us invaluable insight into the genetic basis of cancer [reviewed in Van Dyke and Jacks, 2002]. Oncogenes that are overexpressed or mutated in specific human cancers can also be overexpressed in mice in the same cell type to recapitulate the development of the tumor. For example, the overexpression of myc, which occurs by gene amplification and leads to B-cell lymphomas in humans, also produces B-cell lymphor2003 WILEY-LISS, INC.

mas in mice [Marcu et al., 1992]. Additionally, systems have been developed in mice to turn genes on and off at will [reviewed by Yamamoto et al., 2001]. By manipulating the levels of myc expression using such a system, the importance of myc in maintaining tumorigenesis was unraveled [Felsher and Bishop, 1999]. Upon activation of myc expression, the majority of mice developed T-cell lymphomas (due to the use of a T-cell specific promoter) with a mean latency of about 14 weeks. Subsequent inhibition of myc expression led to tumor regression, suggesting an important role for myc in the maintenance of T-cell lymphomas. Similarly, inducible expression of HrasV12G in mice lacking INK4a yielded melanomas that required ras for tumor maintenance. Decreasing the levels of ras led to tumor reversion [Chin et al., 1999]. These studies suggest that targeting of the myc and ras oncogene products with specific drugs should n Correspondence to: Guillermina Lozano, Ph.D., Department of Molecular Genetics, Box 11, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston,TX 77030 - 4095. E-mail: [email protected]

DOI 10.1002/humu.10186 Published online in Wiley InterScience (www.interscience.wiley. com).

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be sufficient to inhibit tumorigenesis. These experiments, which could never have been done in humans, have yielded invaluable insight into tumorigenesis. THE TP53 NULL MOUSE

Mouse models have also proven extremely useful in studying the mechanism of function of tumor suppressors. Specifically, the generation of mice null for p53 [Donehower et al., 1992; Jacks et al., 1994] has yielded insight into the role of p53 in tumorigenesis and in resistance to chemotherapeutic agents in vivo. Mice missing one or both p53 alleles are predisposed to multiple tumor types such as lymphomas, sarcomas, and brain and lung tumors [Harvey et al., 1993; Jacks et al., 1994]. These resemble the types of tumors seen in patients with Li-Fraumeni syndrome, characterized by the inheritance of a p53 mutation [reviewed by Evans and Lozano, 1997]. Tumors that lacked p53 were resistant to treatment with gamma radiation or adriamycin in contrast to tumors with wild-type p53 that tended to undergo apoptosis readily [Lowe et al., 1994]. Further insight into the mechanism of p53 function was shown when mice developed aggressive tumors in the absence of p53-dependent apoptosis [Symonds et al., 1994]. The kinds of tumors that develop and the timing of occurrence are also dramatically decreased when the p53 null allele is combined with other genetic defects that cooperate in tumorigenesis [reviewed in Lozano and Liu, 1998; Attardi and Jacks, 1999]. The latest example is mice with mutant K-ras induced lung adenocarcinomas that were wild-type or heterozygous for the p53 null allele [Johnson et al., 2001]. Mutations in K-ras combined with loss of one p53 allele showed a significant acceleration in tumor incidence as compared with mice with mutant K-ras alone. TP53 MISSENSE MUTATIONS

More than 50% of human tumors contain p53 mutations, of which 80% are missense mutations mainly found in the DNA-binding domain of p53. These p53 mutants lose the ability to bind DNA and activate transcription and therefore are unable to initiate cell cycle arrest or apoptosis. Tissue culture experiments in which some p53 mutants are overexpressed indicate that missense mutations may represent dominant-negative or gain-of-function phenotypes. The best example is the genesis of tumorigenic potential in cell lines overexpressing the p53R175H mutation as compared to the parental p53 null cells [Dittmer et al., 1993]. Thus, it is possible that the existing p53 null mouse does not recapitulate the tumor incidence of p53 missense mutations. Unfortunately, the analysis of cells in culture also does not provide knowledge as to the role of these mutations in the initiation of tumors, the rate

at which they grow, and the tumor spectrum. These questions can only be addressed by generating in vivo models to assay the function of p53 missense mutations. This review will focus on the contribution of p53 missense mutations to tumorigenesis and on the importance of disrupting the p53 pathway in tumorigenesis. To address these issues in vivo, several investigators are generating mice containing specific p53 missense mutations. One of the goals of generating these models is to compare disease severity between mice lacking p53 and those expressing a mutant p53 protein. Studies of humans to achieve this goal is difficult due to the effects of such variables as nutrition and environment (which are impossible to control) on tumor development. For example, p53 mice on a limited caloric diet live longer than mice fed ad libitum [Berrigan et al., 2002]. Genetic modifiers also exist since we know that different inbred strains of mice carrying the same p53 null allele exhibit differences in the kinds of tumors that develop [Donehower et al., 1995; Muller et al., 2000; Kuperwasser et al., 2000]. Since the most common alteration in p53 both in somatic cells and in the germline of patients with Li Fraumeni syndrome is a missense mutation, it was imperative to develop models more reminiscent of the human condition. The first mouse generated by homologous recombination contains a single nucleotide substitution altering arginine 172 to histidine in the endogenous p53 allele [Liu et al., 2000]. This mutation is equivalent to the arg-to-his substitution at amino acid 175 in human tumors. This mouse contains the additional deletion of a G nucleotide at a splice junction which attenuates levels of mutant p53 to near wild-type levels. Mice heterozygous for the mutant allele (p53R172HDg) differed from p53+/– mice in tumor spectrum, with a significant increase in the number of carcinomas and a slight decrease in the number of lymphomas. More important, the osteosarcomas and carcinomas that developed in these mutant mice frequently metastasized (69% and 40%, respectively). Upon gross observation, numerous white nodules were visible on the surfaces of livers and lungs from these mutant mice. In contrast, micrometastasis is seen in less than 10% of osteosarcomas of p53+/– mice and was absent in carcinomas [Taverna et al., 1998]. These data indicate clear differences between a p53 missense mutation and a null allele in tumorigenesis in vivo and suggest that the p53R172HDg mutant represents a gain-of-function allele. Loss of heterozygosity (LOH) studies using the in situ laser-capture procedure to minimize the presence of normal tissue were also performed on 11 different tumors from p53R172HDg/+ mice of different ages to determine if the wild-type p53 allele had been lost.

Only one of 11 tumors (9%) showed LOH suggesting that loss of wild-type p53 was not important to tumor development. This is in contrast to LOH studies in p53+/– mice where 50% of the mice under 18 months of age were shown to have LOH of the wild-type p53 allele [Venkatachalam et al., 1998]. These data support a dominant negative phenotype for the p53R172HDg allele. In humans, only 47% of tumors analyzed in p53 mutation carriers with missense mutations in the DNA binding domain showed LOH [Birch et al., 1998]. In this study, specific missense mutations could not be analyzed individually owing to the small number of samples. All mutations outside the DNA binding domain and mutations that represent truncations in p53 showed LOH. These studies in humans and in mice indicate that loss of the wild-type p53 is not essential in tumors with a missense mutation in p53. We can extrapolate that the various p53 mutants may have different properties in vivo and the treatment of patients may have to be tailored to the specific p53 mutation. Two other missense mutations mimicking the p53 mutations found in human tumors have been studied in embryonic stem (ES) cells and in thymocytes [de Vries et al., 2002]. These p53 mutants are the arginine-to-histidine alteration at p53 amino acid 270 (corresponding to R273H in humans) and the prolineto-serine mutation at p53 amino acid 275 (corresponding to P278S mutation specific to skin tumors in humans). In ES cells heterozygous for the p53R270H mutation, activation of mdm2, bax, and cyclin G expression was delayed in response to gamma radiation as compared to normal cells. p53P275S heterozygous mutant cells showed markedly reduced expression of mdm2 and bax with a delayed response in cyclin G expression in response to gamma radiation as compared to normal cells. These data suggest that a single copy of either p53 mutant allele can also confer a dominant negative phenotype in ES cells. To further test the dominant negative activity of these mutants, ES cells and thymocytes heterozygous for these mutations were treated with doxorubicin to measure apoptosis. Wild-type cells under these conditions exhibit apoptosis, while p53 null cells do not, and cells heterozygous for the p53 null allele show an intermediate phenotype. In this assay, ES cells with either the p53R270H or the p53P275S mutation in the heterozygous condition showed loss of apoptosis function almost as well as p53 null ES cells. In vivo, apoptosis in thymocytes gave the same results. The experiments in thymocytes were performed by injecting ES cells into Rag-deficient blastocysts. Because Rag-deficient mice cannot make B or T cells, the B and Tcells that appear originate from the manipulated ES cells. This is an easy way to analyze missense mutations without making mice. These data clearly suggest a dominant-negative role for these specific mutations. The generation of mice with these alleles

should provide more insight into the role of these

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endogenous p53 gene with a gene containing a point mutation changing p53 serine 23 (serine 20 in human) to an alanine. This strategy does not overproduce the p53 protein off of a constitutively active promoter, but rather uses the endogenous locus to drive the mutant p53 and so represents a more realistic expression pattern. Homozygous mutant ES cells, MEFs, and thymocytes all show no defect in p53 stability, induction of p53 targets, or induction of apoptosis following DNA damage [Wu et al., 2002]. These data do not suggest that chk2 is not important for p53 stability, but that phosphorylation of p53 serine 20 alone is insufficient to stabilize p53. Activation of p53 in cells from ataxia telangiectasia (AT) patients deficient in the gene ATM is delayed following gamma radiation [Kastan et al., 1992]. Although ATM and a related protein ATR activate chk1 and chk2, they can also directly phosphorylate p53 on ser 15 [reviewed in Appella and Anderson, 2001]. In vitro data suggest two possible nonexclusive mechanisms for the importance of phosphorylation of serine 15. One is, again, that phosphorylation of serine 15 blocks MDM2 binding to p53, and the second is that phosphorylation at serine 15 is a prerequisite for acetylation of the c-terminus of p53 which is required for p53 DNA binding activity. Again ES cells that are homozygous for a mutation at p53 serine 18 (serine 15 in human) to alanine were made [Chao et al., 2000b]. However, in this case the ES cells homozygous for the p53S18A mutation were defective in p53 stabilization and the induction of p53 targets following exposure to ultraviolet light or gamma radiation. In addition, G1 arrest was partially defective following DNA damage. Acetylation of the carboxy terminus was normal in the p53S15A mutant cells, suggesting that acetylation of the C terminus does not depend on phosphorylation of p53 serine 15. Thus, the experiments using ES cells expressing these mutants from the endogenous p53 locus are more representative of the in vivo situation and highlight the importance of p53 serine 15. The analysis of these p53 phosphorylation mutants by generating mice heterozygous and homozygous for the mutation should reveal the in vivo significance of these phosphorylation events. Many additional phosphorylation sites have been described, and mutation of some of these sites in mice is ongoing. In addition, other posttranslational modifications will need to be addressed in vivo to test their importance in regulating p53 function. Ultimately, multiple sites may have to be concertedly modified in vivo to significantly affect p53 function. Disrupting theTP53 Pathway

While missense mutations at specific serines have provided insight into the upstream pathways that regulate p53, two other proteins that regulate p53

function have been identified, MDM2 and MDM4. Again mouse models have provided insight into the importance of these regulatory pathways. The mdm2 gene encodes an E3 ubiquitin ligase that binds and degrades the p53 tumor suppressor [reviewed in Michael and Oren, 2002]. The mdm2 gene is amplified in approximately 30% of sarcomas [Oliner et al., 1992] and is overproduced in a large number of other tumor types [Evans et al., 2001]. Some of these tumors retain wild-type p53, suggesting that MDM2 inactivates p53 in the process of tumorigenesis. In mice, deletion of the mdm2 gene yields an early embryo lethal phenotype [Montes de Oca Luna et al., 1995; Jones et al., 1995]. Since MDM2 inhibits p53 function, the hypothesis that the embryonic lethality seen in embryos lacking mdm2 was due to the inability to down regulate p53 function was tested. Mice lacking mdm2 and p53 were viable and fertile. Thus, the mdm2 loss-of-function phenotype in embryos and in cells is a direct result of a constitutively active p53 and can be used as an in vivo measure of p53 activity. These data further emphasize the role of p53 in early embryogenesis in a normal cell cycle. An MDM2-related protein, MDM4 (originally named MDMX), that has some of the same properties as MDM2 was recently discovered [Shvarts et al., 1996]. MDM4 binds and inhibits p53 transcriptional activity in vitro. However, unlike MDM2, MDM4 does not cause nuclear export or degradation of p53 [Jackson and Berberich, 2000; Stad et al., 2000]. To study MDM4 function in vivo, the mdm4 gene was deleted in mice [Parant et al., 2001; Migliorini et al., 2002; Finch et al., 2002]. In these experiments Mdm4 null mice died during early embryogenesis. Death occurred due to loss of cell proliferation. To examine the importance of p53 in the death of mdm4-deficient embryos, crosses with the p53 null allele were performed. The loss of p53 completely rescued the mdm4 / embryonic lethality. To determine if loss of any two of the mdm alleles resulted in embryo lethality, crosses between mice carrying the mdm2 and mdm4 null alleles were performed. Normal production of progeny heterozygous for both alleles was observed (Parant and Lozano, unpublished observations). Thus, MDM2 and MDM4 are nonoverlapping critical regulators of p53 in vivo. These data define a new pathway of p53 regulation and raises the possibility that increased MDM4 levels and the resulting inactivation of p53 contributes to the development of human tumors. The usefulness of mouse models to study cancer has only begun to be exploited. The ability to make precise alterations such as single nucleotide changes to mimic the defects found in human cancer syndromes will continue to provide insight into our understanding of the genetic basis of cancer. Moreover, risk modifiers can be more easily mapped and

TP53 MOUSE MODELS

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