Sean M. Post

The inherent instability of mutant p53 is alleviated by Mdm2 or p16INK4a loss

The p53 tumor suppressor is often disrupted in human cancers by the acquisition of missense mutations. We generated mice with a missense mutation at codon 172 that mimics the p53R175H hot spot mutation in human cancer. p53 homozygous mutant mice have unstable mutant p53 in normal cells and stabilize mutant p53 in some but not all tumors. To investigate the significance of these data, we examined the regulation of mutant p53 stability by Mdm2, an E3 ubiquitin ligase that targets p53 for degradation, and p16INK4a, a member of the Rb tumor suppressor pathway. Mice lacking Mdm2 or p16INK4a stabilized mutant p53, and revealed an earlier age of tumor onset than p53 mutant mice and a gain-of-function metastatic phenotype. Analysis of tumors from p53 homozygous mutant mice with stable p53 revealed defects in the Rb pathway. Additionally, ionizing radiation stabilizes wild-type and mutant p53. Thus, the stabilization of mutant p53 is not a given but it is a prerequisite for its gain-of-function phenotype. Since mutant p53 stability mimics that of wild-type p53, these data indicate that drugs aimed at activating wild-type p53 will also stabilize mutant p53 with dire consequences.

A High-Frequency Regulatory Polymorphism in the p53 Pathway Accelerates Tumor Development

MDM2, a negative regulator of p53, is elevated in many cancers that retain wild-type p53. A single nucleotide polymorphism (SNP) in the human MDM2 promoter increases the affinity of Sp1 resulting in elevated MDM2 levels. We generated mice carrying either the MDM2(SNP309T) or the MDM2(SNP309G) allele to address the impact of MDM2(SNP309G) on tumorigenesis. Mdm2(SNP309G/G) cells exhibit elevated Mdm2 levels, reduced p53 levels, and decreased apoptosis. Importantly, some Mdm2(SNP309G/G) mice succumbed to tumors before 1 year of age, suggesting that this allele increases tumor risk. Additionally, the Mdm2(SNP309G) allele potentiates the tumor phenotype and alters tumor spectrum in mice inheriting a p53 hot-spot mutation. These data provide causal evidence for increased cancer risk in carriers of the Mdm2(SNP309G) allele.

Multiple stress signals activate mutant p53 in vivo

p53 levels are tightly regulated in normal cells, and thus, the wild-type p53 protein is nearly undetectable until stimulated through a variety of stresses. In response to stress, p53 is released from its negative regulators, mainly murine double minute 2 (Mdm2), allowing p53 to be stabilized to activate cell-cycle arrest, senescence, and apoptosis programs. Many of the upstream signals that regulate wild-type p53 are known; however, limited information for the regulation of mutant p53 exists. Previously, we showed that wild-type and mutant p53R172H are regulated in a similar manner in the absence of Mdm2 or p16. In addition, this stabilization of mutant p53 is responsible for the gain-of-function metastatic phenotype observed in the mouse. In this report, we examined the role of oncogenes, DNA damage, and reactive oxygen species, signals that stabilize wild-type p53, on the stabilization of mutant p53 in vivo and the consequences of this expression on tumor formation and survival. These factors stabilized mutant p53 protein which oftentimes contributed to exacerbated tumor phenotypes. These findings, coupled with the fact that patients carry p53 mutations without stabilization of p53, suggest that personalized therapeutic schemes may be needed for individual patients depending on their p53 status.

Spontaneous tumorigenesis in mice overexpressing the p53-negative regulator Mdm4.

High levels of the critical p53 inhibitor Mdm4 is common in tumors that retain a wild-type p53 allele, suggesting that Mdm4 overexpression is an important mechanism for p53 inactivation during tumorigenesis. To test this hypothesis in vivo, we generated transgenic mice with widespread expression of Mdm4. Two independent lines of transgenic mice, Mdm4(Tg1) and Mdm4(Tg15), developed spontaneous tumors, the most prevalent of which were sarcomas. To determine whether overexpression of Mdm4 also cooperated with p53 heterozygosity to induce tumorigenesis, we generated Mdm4(Tg1) p53(+/-) mice. These mice had significantly accelerated tumorigenesis and a distinct tumor spectrum with more carcinomas and significantly fewer lymphomas than p53(+/-) or Mdm4(Tg1) mice. Importantly, the remaining wild-type p53 allele was retained in most Mdm4(Tg1) p53(+/-) tumors. Mdm4 is thus a bona fide oncogene in vivo and cooperates with p53 heterozygosity to drive tumorigenesis. These Mdm4 mice will be invaluable for in vivo drug studies of Mdm4 inhibitors.

p53-dependent senescence delays Eμ-myc- induced B-cell lymphomagenesis

The effect of p53-dependent cell-cycle arrest and senescence on Eμ-myc-induced B-cell lymphoma development remains controversial. To address this question, we crossed Eμ-myc mice with the p53515C mutant mouse, encoding the mutant p53R172P protein that retains the ability to activate the cell-cycle inhibitor and senescence activator p21. Importantly, this mutant lacks the ability to activate p53-dependent apoptotic genes. Hence, Eμ-myc mice that harbor two p53515C alleles are completely defective for p53-dependent apoptosis. Both Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice survive significantly longer than Eμ-myc::p53+/– mice, indicating the importance of the p53-dependent non-apoptotic pathways in B-cell lymphomagenesis. In addition, the p53515C allele is deleted in several Eμ-myc::p53515C/+ lymphomas, further emphasizing the functionality of p53R172P in tumor inhibition. Lymphomas from both Eμ-myc::p53515C/515C and Eμ-myc::p53515C/+ mice retain the ability to upregulate p21, resulting in cellular senescence. Senescence-associated β-galactosidase (SA β-gal) activity was observed in lymphomas from Eμ-myc::p53+/+, Eμ-myc::p53515C/515C and Eμ-myc::p53515C /+ mice but not in lymphomas isolated from Eμ-myc::p53+/– mice. Thus, in the absence of p53-dependent apoptosis, the ability of p53R172P to induce senescence leads to a significant delay in B-cell lymphoma development.

Regulation of tissue- and stimulus-specific cell fate decisions by p53 in vivo

The tumour suppressor p53 pathway is often inactivated by multiple mechanisms in the genesis of human cancers. Aberrant cellular proliferation, DNA damage, hypoxia, and ribosomal stress cause activation of the p53 tumour suppressor with multiple possible consequences to the cell: cell death, cell cycle arrest, or senescence. These mechanisms ultimately ensure that the cell does not replicate, and are thus potent tumour suppressor mechanisms. An important question that has eluded the field is how p53 makes these cell fate decisions. This review summarizes the current status of knowledge regarding p53‐mediated stress and tissue‐dependent cell fate decisions in mouse models and human tumours. Copyright

E2F3 is a mediator of DNA damage-induced apoptosis.

ABSTRACT The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member E2F3a can be induced by DNA damage through transcriptional and posttranslational mechanisms. We demonstrate that the posttranslational induction of human E2F3a is dependent on the checkpoint kinases. Moreover, we show that human E2F3a is a substrate for the checkpoint kinases (chk kinases) and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that E2F1 and E2F2 are transcriptionally induced by DNA damage in an E2f3-dependent manner. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage-induced apoptosis. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.

The p53–Mdm2 feedback loop protects against DNA damage by inhibiting p53 activity but is dispensable for p53 stability, development, and longevity

The p53-Mdm2 feedback loop is perceived to be critical for regulating stress-induced p53 activity and levels. However, this has never been tested in vivo. Using a genetically engineered mouse with mutated p53 response elements in the Mdm2 P2 promoter, we show that feedback loop-deficient Mdm2(P2/P2) mice are viable and aphenotypic and age normally. p53 degradation kinetics after DNA damage in radiosensitive tissues remains similar to wild-type controls. Nonetheless, DNA damage response is elevated in Mdm2(P2/P2) mice. Enhanced p53-dependent apoptosis sensitizes hematopoietic stem cells (HSCs), causing drastic myeloablation and lethality. These results suggest that while basal Mdm2 levels are sufficient to regulate p53 in most tissues under homeostatic conditions, the p53-Mdm2 feedback loop is critical for regulating p53 activity and sustaining HSC function after DNA damage. Therefore, transient disruption of p53-Mdm2 interaction could be explored as a potential adjuvant/therapeutic strategy for targeting stem cells in hematological malignancies.

Effects of MDM2, MDM4 and TP53 codon 72 polymorphisms on cancer risk in a cohort study of carriers of TP53 germline mutations.

Background Previous studies have shown that MDM2 SNP309 and p53 codon 72 have modifier effects on germline P53 mutations, but those studies relied on case-only studies with small sample sizes. The impact of MDM4 polymorphism on tumor onset in germline mutation carriers has not previously been studied. Methodology/Principal Findings We analyzed 213 p53 germline mutation carriers including 168(78.9%) affected with cancer and 174 who had genotypic data. We analyzed time to first cancer using Kaplan-Meier and Cox proportional hazards methods, comparing risks according to polymorphism genotypes. For MDM2 SNP309, a significant difference of 9.0 years in the average age of cancer diagnosis was observed between GG/GT and TT carriers (18.6 versus 27.6 years, P = 0.0087). The hazards ratio was 1.58 (P = 0.03) comparing risks among individuals with GG/GT to risk among TT, but this effect was only significant in females (HR = 1.60, P = 0.02). Compared to other genotypes, P53 codon 72 PP homozygotes had a 2.24 times (P = 0.03) higher rate for time to develop cancer. We observed a multiplicative joint effect of MDM2 and p53 codon72 polymorphism on risk. The MDM4 polymorphism had no significant effects. Conclusions/Significance Our results suggest that the MDM2 SNP309 G allele is associated with cancer risk in p53 germline mutation carriers and accelerates time to cancer onset with a pronounced effect in females. A multiplicative joint effect exists between the MDM2 SNP309 G allele and the p53 codon 72 G allele in the risk of cancer development. Our results further define cancer risk in carriers of germline p53 mutations.

Role of neoplastic monocyte-derived fibrocytes in primary myelofibrosis.

Estrov and collaborators examine the role of fibrocytes in primary myelofibrosis and propose a novel therapeutic approach.