The p53 protein integrates multiple upstream signals and functions as a tumor suppressor by activating distinct downstream genes 1-3 . At the cellular level, p53 induces apoptosis, cell cycle arrest and senescence. A rare mutant form of p53 with the amino acid substitution R175P, found in human tumors, is completely defective in initiating apoptosis but still induces cell cycle arrest 4,5 . To decipher the functional importance of these pathways in spontaneous tumorigenesis, we used homologous recombination to generate mice with mutant p53-R172P (the mouse equivalent of R175P in humans). Mice inheriting two copies of this mutation (Trp53 515C/515C ) escape the early onset of thymic lymphomas that characterize Trp53-null mice. At 7 months of age, 90% of Trp53-null mice had died, but 85% of Trp53 515C/515C mice were alive and tumor-free, indicating that p53-dependent apoptosis was not required for suppression of early onset of spontaneous tumors. The lymphomas and sarcomas that eventually developed in Trp53 515C/515C mice retained a diploid chromosome number, in sharp contrast to aneuploidy observed in tumors and cells from Trp53-null mice. The ability of mutant p53-R172P to induce a partial cell cycle arrest and retain chromosome stability are crucial for suppression of early onset tumorigenesis.We introduced the 515G→C mutation into the Trp53 locus by homologous recombination into embryonic stem (ES) cells and cre-loxP-mediated excision in mice (Fig. 1). Sequencing of the entire coding region of the Trp53 transcript from a homozygous mutant mouse found no other changes. The allele Trp53 515C expressed a fulllength mutant p53 protein with the amino acid substitution R172P, which was more abundant than wild-type p53 (Fig. 1d).To assay the cell cycle arrest function, we treated subconfluent cultures of wild-type, Trp53-null, and Trp53 515C/515C mouse embryonic fibroblasts (MEFs) with γ-radiation and labeled them with 5-bromo-deoxyuridine (BrdU) to measure the number of cells in S phase 6 . The ratio of cells in S phase among cells treated with γ-radiation versus untreated cells was significantly lower in Trp53 515C/515C MEFs than in Trp53-null MEFs (Fig. 2a), but the

p53 R172H/+ mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53 +/− mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53 R172H/+ mice with metastasis to osteosarcomas from p53 +/− mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53 R172H/+ osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformationspecific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis. mammary tumor | fatty acid metabolism T he p53 tumor suppressor pathway is inactivated in ∼50% of human cancers (http://p53.iarc.fr). Missense mutations in particular account for 80% of p53 alterations, suggesting that mutant p53 proteins provide additional advantages for tumor cell growth (1). Li-Fraumeni syndrome patients with p53 missense mutations have a higher cancer incidence and an earlier age of tumor onset than individuals with truncating or splicing mutations (2). p53 knockin mice show a gain-of-function (GOF) phenotype in vivo, with high metastatic capacity compared with mice inheriting a p53-null allele (3, 4). GOF activities of mutant p53 are mediated by suppression of the p53 family members, p63 and p73 (3-6). Other mechanisms of mutant p53 GOF include mutant-p53 complexes with Smad that fuel TGF-β-induced metastasis (7) and integrin recycling (8). Additionally, mutant p53 interacts with the vitamin D receptor and converts vitamin D into an antiapoptotic agent (9-14). More recently, mutant p53 was reported to form transcriptional complexes on promoters of genes encoding several enzymes of the Mevalonate pathway, which increases metastasis of breast cancer cells (9). These data suggest multiple pathways contribute to the GOF phenotypes of cells with mutant p53. Although mutant p53 lacks sequencespecific DNA binding activity, its interaction with other transcriptional factors or the components of basic transcriptional machinery allow it to modulate gene expression (15). ChIP-onchip and ChIP-sequencing techniques show that mutant p53 affects transcription of many genes (9, 13, 16, 17).In this study, expression array analyses identified gene differences between p53 R172H/+ m...

Mdm2 inhibits the function of the p53 tumor suppressor. Mdm2 is overexpressed in many tumors with wild-type p53 suggesting an alternate mechanism of loss of p53 activity in tumors. An Mdm2-binding protein (MTBP) was identified using a yeast two-hybrid screen. In tissue culture, MTBP inhibits Mdm2 self-ubiquitination, leading to stabilization of Mdm2 and increased degradation of p53. To address the role of MTBP in the regulation of the p53 pathway in vivo, we deleted the Mtbp gene in mice. Homozygous disruption of Mtbp resulted in early embryonic lethality, which was not rescued by loss of p53. Mtbp þ /À mice were not tumor prone. When mice were sensitized for tumor development by p53 heterozygosity, we found that the Mtbp þ /À p53 Introduction p53 is a transcription factor that activates numerous downstream genes with roles in cell cycle arrest, apoptosis, DNA repair and senescence (Levine et al., 2004). As such, disruption of the p53 pathway is a critical event in human cancer. Mutations or deletions in the p53 gene occur in approximately 50% of cancers (Bartek et al., 1991;Levine, 1993;Vogelstein et al., 2000). The p53 pathway is also inactivated by overexpression of the p53 inhibitor, Mdm2. Overexpression of Mdm2 by several mechanisms occurs in about 30% of sarcomas and many other types of tumors (Iwakuma and Lozano, 2003). ARF (alternative reading frame of the Ink 4a locus), another important regulator of this pathway, binds and inhibits Mdm2 by relocating Mdm2 from nucleus to nucleolus (Sharpless and DePinho, 1999;Weber et al., 1999). Therefore, ARF loss in tumors releases Mdm2, leading to increased binding to and inactivation of p53. Thus, the ARF-Mdm2-p53 pathway is a key pathway that must be bypassed in tumor development.Experiments using genetically engineered mice have provided direct evidence for the importance of these factors in tumorigenesis. Mice with homozygous or heterozygous deletion of p53 develop tumors (Donehower et al., 1992;Tsukada et al., 1993;Jacks et al., 1994;Purdie et al., 1994). Mice with missense mutations identical to those found in humans with Li-Fraumeni syndrome develop a variety of tumors with frequent metastasis (Lang et al., 2004;Olive et al., 2004 MTBP was isolated as a novel Mdm2-binding protein (Boyd et al., 2000). The MTBP-Mdm2 interaction was observed using purified proteins in vitro and by overexpression in cells. Overexpression of MTBP protects Mdm2 from self-ubiquitination, leading to Mdm2 stabilization and p53 degradation (Brady et al., 2005). We therefore hypothesized that MTBP affects tumor development through modulation of p53 activity. To test this hypothesis and examine the in vivo physiological function of MTBP, we generated mice with disruption of the Mtbp gene. Our results show that Mtbp-null mice were embryonic lethal, and this phenotype was p53 independent. Mtbp þ /À mice were normal, but when crossed with p53 þ /À mice, Mtbpþ /À mice developed significantly more metastatic tumors compared to p53 þ /À mice. In vitro invasion assays clearly supported the in ...

The p53 tumor suppressor is mutated in the majority of human tumors. MDM2, a well-known inhibitor of p53, is overexpressed in a large number of tumors suggesting that increased levels of MDM2 also contribute to tumorigenesis. A novel p53 inhibitor, MDM4, was more recently identified. The role of MDM4 in cancer development is not well understood. We set out to examine the levels of MDM4 by immunohistochemistry in Head and Neck Squamous Carcinomas (HNSC) to ask whether high MDM4 levels could contribute to its development and progression. In addition, MDM2 and p53 levels were examined to identify overlapping expression patterns. MDM4 is present at high levels in 50% of HNSC. Additionally, overexpression of MDM2 was detected in 80% of tumors, many of which were also positive for MDM4. A subset of tumors displayed high levels of all three proteins. Sequencing of the p53 gene revealed that tumors with positive immunoreactivity for MDM2 or MDM4, some of which also had high levels of p53, did not carry mutations in this gene. Thus, the detection of p53 by immunohistochemistry was not synonymous with the presence of p53 mutations. Expression of both MDM2 and MDM4 in tumors without p53 mutations strongly suggests that MDM2 and MDM4 inhibit the activity of this tumor suppressor in HNSC.

TP53 mutations are the most frequent genetic alterations in breast cancer and are associated with more aggressive disease and worse overall survival. We have created two conditional mutant Trp53 alleles in the mouse that allow expression of Trp53R172H or Trp53R245W missense mutations in single cells surrounded by a normal stroma and immune system. Mice with Trp53 mutations in a few breast epithelial cells develop breast cancers with high similarity to human breast cancer including triple negative. p53R245W tumors are the most aggressive and exhibit metastases to lung and liver. Development of p53R172H breast tumors with some metastases requires additional hits. Sequencing of primary tumors and metastases shows p53R245W drives a parallel evolutionary pattern of metastases. These in vivo models most closely simulate the genesis of human breast cancer and will thus be invaluable in testing novel therapeutic options.

Tumor sequencing studies have emphasized the role of epigenetics and altered chromatin homeostasis in cancer. Mutations in DAXX, which encodes a chaperone for the histone 3.3 variant, occur in 25% of pancreatic neuroendocrine tumors (PanNETs). To advance our understanding of physiological functions of Daxx, we developed a conditional Daxx allele in mice. We demonstrate that Daxx loss is well tolerated in the pancreas but creates a permissive transcriptional state that cooperates with environmental stress (inflammation) and other genetic lesions (Men1 loss) to alter gene expression and cell state, impairing pancreas recovery from inflammatory stress in vivo. The transcriptional changes are associated with dysregulation of endogenous retroviral elements (ERVs), and dysregulation of endogenous genes near ERVs is also observed in human PanNETs with DAXX mutations. Our results reveal a physiologic function of DAXX, provide a mechanism associated with impaired tissue regeneration and tumorigenesis, and expand our understanding of ERV regulation in somatic cells.

The p53 (TP53) tumor suppressor is the most frequently mutated gene in human cancers. Restoring expression of wild-type p53 has led to tumor growth suppression in a variety of tumor models that are p53 deficient. Other mechanisms, e.g. up-regulation of Mdm2, exist in tumors to inactivate the p53 pathway. Mdm2, an E3 ubiquitin-ligase that targets p53 for proteasomal degradation, is present at high levels in many tumors with wild-type p53. In this study, the effects of restoring p53 activity were probed in Mdm2-overexpressing tumors genetically using animal models. Here it was demonstrated that elevated levels of Mdm2 and decreased levels of p53 act additively to dampen p53 activity in DNA damage response and tumor development. Our data further indicate that restoration of wild-type p53 expression in Mdm2-overexpressing angiosarcomas results in tumor stasis and regression in some cases. Finally, it was determined that restored p53 suppressed cell proliferation but did not elicit apoptosis in the Mdm2-overexpressing angiosarcomas.

Gastrointestinal (GI) syndrome is a serious side effect and dose-limiting toxicity observed in patients undergoing lower-abdominal radiotherapy. Previous mouse studies show that p53 gene dosage determines susceptibility to GI syndrome development. However, the translational relevance of p53 activity has not been addressed. Here, we used a knock-in mouse in which the p53–Mdm2 negative feedback loop is genetically disrupted. These mice retain biallelic p53 and thus, normal basal p53 levels and activity. However, due to the lack of p53-mediated Mdm2 transcription, irradiated Mdm2P2/P2 mice exhibit enhanced acute p53 activity, which protects them from GI failure. Intestinal crypt cells residing in the +4 and higher positions exhibit decreased apoptosis, increased p21 expression, and hyperproliferation to reinstate intestinal integrity. Correspondingly, pharmacological augmentation of p53 activity in wild-type mice with an Mdm2 inhibitor protects against GI toxicity without affecting therapeutic outcome. Our results suggest that transient disruption of the p53–Mdm2 interaction to enhance p53 activity could be a viable prophylactic strategy for alleviating GI syndrome in patients undergoing radiotherapy.