Dimeric p53 Mutant Elicits Unique Tumor-Suppressive Activities through an Altered Metabolic Program.

Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li-Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies. SIGNIFICANCE: New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li-Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are "basal" p53 activities. See related commentary by Stieg et al., p. 1046. See related article by Choe et al., p. 1250. This article is highlighted in the In This Issue feature, p. 1027.

Tissue specificity and spatio-temporal dynamics of the p53 transcriptional program.

Transcription factors regulate hundreds of genes and p53 is no exception. As a stress responsive protein, p53 transactivates an array of downstream targets which define its role in maintaining physiological functions of cells/tissues. Despite decades of studies, our understanding of the p53 in vivo transcriptional program is still incomplete. Here we discuss some of the physiological stressors that activate p53, the pathological and physiological implications of p53 activation and the molecular profiling of the p53 transcriptional program in maintaining tissue homeostasis. We argue that the p53 transcriptional program is spatiotemporally regulated in a tissue-specific manner and define a p53 target signature that faithfully depicts p53 activity. We further emphasize that additional in vivo studies are needed to refine the p53 transactivation profile to harness it for therapeutic purposes.

Differential Gain-of-Function Activity of Three p53 Hotspot Mutants In Vivo.

The majority of TP53 missense mutations identified in cancer patients are in the DNA-binding domain and are characterized as either structural or contact mutations. These missense mutations exhibit inhibitory effects on wild-type p53 activity. More importantly, these mutations also demonstrate gain-of-function (GOF) activities characterized by increased metastasis, poor prognosis, and drug resistance. To better understand the activities by which TP53 mutations, identified in Li-Fraumeni syndrome, contribute to tumorigenesis, we generated mice harboring a novel germline Trp53R245W allele (contact mutation) and compared them with existing models with Trp53R172H (structural mutation) and Trp53R270H (contact mutation) alleles. Thymocytes from heterozygous mice showed that all three hotspot mutations exhibited similar inhibitory effects on wild-type p53 transcription in vivo, and tumors from these mice had similar levels of loss of heterozygosity. However, the overall survival of Trp53R245W/+ and Trp53R270H/+ mice, but not Trp53R172H/+ mice, was significantly shorter than that of Trp53+/- mice, providing strong evidence for p53-mutant-specific GOF contributions to tumor development. Furthermore, Trp53R245W/+ and Trp53R270H/+ mice had more osteosarcoma metastases than Trp53R172H/+ mice, suggesting that these two contact mutants have stronger GOF in driving osteosarcoma metastasis. Transcriptomic analyses using RNA sequencing data from Trp53R172H/+, Trp53R245W/+, and Trp53R270H/+ primary osteosarcomas in comparison with Trp53+/- indicated that GOF of the three mutants was mediated by distinct pathways. Thus, both the inhibitory effect of mutant over wild-type p53 and GOF activities of mutant p53 contributed to tumorigenesis in vivo. Targeting p53 mutant-specific pathways may be important for therapeutic outcomes in osteosarcoma. SIGNIFICANCE: p53 hotspot mutants inhibit wild-type p53 similarly but differ in their GOF activities, with stronger tumor-promoting activity in contact mutants and distinct protein partners of each mutant driving tumorigenesis and metastasis.

Alterations of the Mdm2 C-Terminus Differentially Impact Its Function In Vivo.

Murine double minute 2 (Mdm2) is the principal E3-ubiquitin ligase for p53 and contains a C2H2C4 type RING domain wherein the last cysteine residue is followed by an evolutionarily conserved 13 amino acid C-terminal tail. Previous studies have indicated that integrity of the C-terminal tail is critical for Mdm2 function. Recently, a mutation extending the MDM2 length by five amino acids was identified and associated with enhanced p53 response in fibroblasts and premature aging in a human patient. To investigate the importance of the conserved Mdm2 C-terminal length on p53 regulatory function in vivo, we engineered three novel mouse alleles using CRISPR-Cas9 technology. Genetic studies with these murine models showed that curtailing Mdm2 C-terminal length by even a single amino acid leads to p53-dependent embryonic lethality. Extension of the Mdm2 C-terminal length by five amino acids (QLTCL) yielded viable mice that are smaller in size, exhibit fertility problems, and have a shortened life span. Analysis of early passage mouse embryonic fibroblasts indicated impaired Mdm2 function correlates with enhanced p53 activity under stress conditions. Furthermore, analysis in mice showed tissue-specific alterations in p53 target gene expression and enhanced radiosensitivity. These results confirm the physiological importance of the evolutionarily conserved Mdm2 C-terminus in regulating p53 functions. SIGNIFICANCE: This in vivo study highlights that alterations to the C-terminus of Mdm2 perturb its regulation of the tumor suppressor p53.

Transient enhancement of p53 activity protects from radiation-induced gastrointestinal toxicity.

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.

Dicer1 Phosphomimetic Promotes Tumor Progression and Dissemination.

Dicer1 functions as a tumor suppressor in mouse models. In humans, somatic mutations are associated with many cancers in adults, and patients with DICER1 syndrome with DICER1 germline mutations are susceptible to childhood cancers. Dicer is phosphorylated by the ERK-MAP kinase pathway and because this pathway is activated in human cancers, we asked whether phosphorylated Dicer1 contributed to tumor development. In human endometrioid cancers, we discovered that phosphorylated DICER1 is significantly associated with invasive disease. To test a direct involvement of Dicer1 phosphorylation in tumor development, we studied mice with phosphomimetic alterations at the two conserved serines phosphorylated by ERK and discovered that a phosphomimetic Dicer1 drives tumor development and dissemination in two independent murine cancer models (KRas+/LA1 and p53+/- ). Our findings demonstrate that phosphomimetic Dicer1 promotes tumor development and invasion. SIGNIFICANCE: This work highlights the relevance of Dicer1 phosphorylation in mammalian tumor development and dissemination.

Constitutive Dicer1 phosphorylation accelerates metabolism and aging in vivo.

DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.

Somatic Trp53 mutations differentially drive breast cancer and evolution of metastases.

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.

Daxx Functions Are p53-Independent In Vivo.

Mutations in the death domain-associated protein (DAXX) have been recently identified in a substantial proportion of human pancreatic neuroendocrine tumors (PanNETs). Remarkably, however, little is known about the physiologic role(s) of DAXX despite in vitro studies suggesting potential functions. Most prominently, and supported by tumor sequencing data, DAXX functions in concert with alpha thalassemia/mental retardation X-linked (ATRX) as a histone chaperone complex for the H3.3 variant. Studies have also identified potential roles in apoptosis, transcription, and negative regulation of the p53 tumor suppressor pathway. Herein, a mouse modeling approach was used to specifically address the latter and no significant genetic interaction between Daxx and the p53 pathway was determined. The embryonic lethal phenotype of Daxx loss is not p53-dependent. In addition, Daxx heterozygosity does not sensitize mice to a sublethal dose of ionizing radiation or alter the survival or tumor phenotype of Mdm2 transgenic mice. However, the data support a tumor suppressor role for DAXX as low-dose ionizing radiation produced a higher proportion of carcinomas in Daxx heterozygous mice than wild-type controls.Implications: While DAXX has important in vivo functions, they are independent of an inhibitory role on the p53 tumor suppressor pathway. Mol Cancer Res; 16(10); 1523-9. ©2018 AACR.

Synergistic and additive effect of retinoic acid in circumventing resistance to p53 restoration.

TP53 mutations occur in ∼50% of all human tumors, with increased frequency in aggressive cancers that are notoriously difficult to treat. Additionally, p53 missense mutations are remarkably predictive of refractoriness to chemo/radiotherapy in various malignancies. These observations have led to the development of mutant p53-targeting agents that restore p53 function. An important unknown is which p53-mutant tumors will respond to p53 reactivation-based therapies. Here, we found a heterogeneous impact on therapeutic response to p53 restoration, suggesting that it will unlikely be effective as a monotherapy. Through gene expression profiling of p53R172H -mutant lymphomas, we identified retinoic acid receptor gamma (RARγ) as an actionable target and demonstrated that pharmacological activation of RARγ with a synthetic retinoid sensitizes resistant p53-mutant lymphomas to p53 restoration, while additively improving outcome and survival in inherently sensitive tumors.

Loss of digestive organ expansion factor (Diexf) reveals an essential role during murine embryonic development that is independent of p53.

Increased levels of inhibitors of the p53 tumor suppressor such as Mdm2 and Mdm4 drive tumor development and thus serve as targets for therapeutic intervention. Recently, digestive organ expansion factor (Diexf) has been identified as a novel inhibitor of p53 in zebrafish. Here, we address the potential role of Diexf as a regulator of the p53 pathway in mammals by generating Diexf knockout mice. We demonstrate that, similar to Mdm2 and Mdm4, homozygous deletion of Diexf is embryonic lethal. However, unlike in Mdm2 and Mdm4 mice, loss of p53 does not rescue this phenotype. Moreover, Diexf heterozygous animals are not sensitive to sub-lethal ionizing radiation. Thus, we conclude that Diexf is an essential developmental gene in the mouse, but is not a significant regulator of the p53 pathway during development or in response to ionizing radiation.

Tumorigenesis promotes Mdm4-S overexpression.

Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and Mdm4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of Mdm4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of Mdm4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that Mdm4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution.

The p53 inhibitor Mdm4 cooperates with multiple genetic lesions in tumourigenesis.

The p53 inhibitor Mdm4 is present at high levels in multiple human cancers. Overexpression of Mdm4 in mice drives the spontaneous development of mostly lymphomas and sarcomas. In this study, we explored the ability of Mdm4 to cooperate with lesions in tumour development. The Mdm4 transgene contributed to mammary tumour development in a BALB/cJ background. High levels of Mdm4 enhanced tumour development in a mutant p53R172H heterozygous background, and reduced the need to lose the wild-type p53 allele, as compared with mice heterozygous only for the p53R172H mutation. Additionally, high levels of Mdm4 cooperated with an oncogenic K-ras mutation to drive lung tumourigenesis in vivo. Finally, we examined p53-independent functions of Mdm4 by studying the contribution of Mdm4 to tumour development in the absence of p53. Whereas the overall survival times of p53-null mice with and without the Mdm4 transgene were similar, male mice with both alterations showed significantly shorter survival than p53-null male mice, and showed differences in tumour spectrum, demonstrating a p53-independent function of Mdm4 in tumourigenesis. Furthermore, p53-null mice with the highest level of Mdm4 tended to have multiple tumours. Thus, a detailed analysis of Mdm4 transgenic mice in various genetic backgrounds shows synergy in tumour development in vivo. Mdm4 may thus serve as a therapeutic target in cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

TRIM24 suppresses development of spontaneous hepatic lipid accumulation and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Aberrantly high expression of TRIM24 occurs in human cancers, including hepatocellular carcinoma. In contrast, TRIM24 in the mouse is reportedly a liver-specific tumour suppressor. To address this dichotomy and to uncover direct regulatory functions of TRIM24 in vivo, we developed a new mouse model that lacks expression of all Trim24 isoforms, as the previous model expressed normal levels of Trim24 lacking only exon 4. METHODS: To produce germline-deleted Trim24(dlE1) mice, deletion of the promoter and exon 1 of Trim24 was induced in Trim24(LoxP) mice by crossing with a zona pellucida 3-Cre line for global deletion. Liver-specific deletion (Trim24(hep)) was achieved by crossing with an albumin-Cre line. Phenotypic analyses were complemented by protein, gene-specific and global RNA expression analyses and quantitative chromatin immunoprecipitation. RESULTS: Global loss of Trim24 disrupted hepatic homeostasis in 100% of mice with highly significant, decreased expression of oxidation/reduction, steroid, fatty acid, and lipid metabolism genes, as well as increased expression of genes involved in unfolded protein response, endoplasmic reticulum stress and cell cycle pathways. Trim24(dlE1/dlE1) mice have markedly depleted visceral fat and, like Trim24(hep/hep) mice, spontaneously develop hepatic lipid-filled lesions, steatosis, hepatic injury, fibrosis and hepatocellular carcinoma. CONCLUSIONS: TRIM24, an epigenetic co-regulator of transcription, directly and indirectly represses hepatic lipid accumulation, inflammation, fibrosis and damage in the murine liver. Complete loss of Trim24 offers a model of human non-alcoholic fatty liver disease, steatosis, fibrosis and development of hepatocellular carcinoma in the absence of high-fat diet or obesity.

Limiting the power of p53 through the ubiquitin proteasome pathway.

The ubiquitin proteasome pathway is critical in restraining the activities of the p53 tumor suppressor. Numerous E3 and E4 ligases regulate p53 levels. Additionally, deubquitinating enzymes that modify p53 directly or indirectly also impact p53 function. When alterations of these proteins result in increased p53 activity, cells arrest in the cell cycle, senesce, or apoptose. On the other hand, alterations that result in decreased p53 levels yield tumor-prone phenotypes. This review focuses on the physiological relevance of these important regulators of p53 and their therapeutic implications.

Pla2g16 phospholipase mediates gain-of-function activities of mutant p53.

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 transformation-specific (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.

Dissecting the p53-Mdm2 feedback loop in vivo: uncoupling the role in p53 stability and activity.

The p53-Mdm2 feedback loop is thought to be the main mechanism by which p53 autoregulates its levels and activity after DNA damage. We tested this paradigm in a genetically engineered mouse model in which the feedback loop was disrupted by point mutations in the p53 binding site of the Mdm2 promoter. We noted that while the p53-Mdm2 feedback loop is required to regulate p53 activity especially in the hematopoietic system in response to DNA damage, its role in development and in regulating the stability of p53 is dispensable. In the present study we have extended our characterization of this mouse model and show that the kinetics of p53 degradation is also unchanged in mouse embryonic fibroblasts (MEFs). Additionally, MG132 experiments indicate that other E3-ligases regulate p53 stability. Also, Mdm4 cooperates in inhibition of p53 activity and levels in these mice. Finally, we show in this system that enhanced acute p53 response does not promote aging or protect against late term tumorigenesis. We also discuss future perspectives for this study.

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.

The p53 pathway in hematopoiesis: lessons from mouse models, implications for humans.

Aberrations in the p53 tumor suppressor pathway are associated with hematologic malignancies. p53-dependent cell cycle control, senescence, and apoptosis functions are actively involved in maintaining hematopoietic homeostasis under normal and stress conditions. Whereas loss of p53 function promotes leukemia and lymphoma development in humans and mice, increased p53 activity inhibits hematopoietic stem cell function and results in myelodysplasia. Thus, exquisite regulation of p53 activity is critical for homeostasis. Most of our understanding of p53 function in hematopoiesis is derived from genetically engineered mice. Here we summarize some of these models, the various mechanisms that disrupt the regulation of p53 activity, and their relevance to human disease.

p53-mediated senescence impairs the apoptotic response to chemotherapy and clinical outcome in breast cancer.

Studies on the role of TP53 mutation in breast cancer response to chemotherapy are conflicting. Here, we show that, contrary to dogma, MMTV-Wnt1 mammary tumors with mutant p53 exhibited a superior clinical response compared to tumors with wild-type p53. Doxorubicin-treated p53 mutant tumors failed to arrest proliferation, leading to abnormal mitoses and cell death, whereas p53 wild-type tumors arrested, avoiding mitotic catastrophe. Senescent tumor cells persisted, secreting senescence-associated cytokines exhibiting autocrine/paracrine activity and mitogenic potential. Wild-type p53 still mediated arrest and inhibited drug response even in the context of heterozygous p53 point mutations or absence of p21. Thus, we show that wild-type p53 activity hinders chemotherapy response and demonstrate the need to reassess the paradigm for p53 in cancer therapy.