Omics analyses of a somatic Trp53R245W/+ breast cancer model identify cooperating driver events activating PI3K/AKT/mTOR signaling.

Alterations of the tumor suppressor TP53, one of the most common events in cancer, alone are insufficient for tumor development but serve as drivers of transformation. We sought to identify cooperating events through genomic analyses of a somatic Trp53R245W mouse model (equivalent to the TP53R248W hot spot mutation in human cancers) that recapitulates metastatic breast-cancer development. We identified cooperating lesions similar to those found in human breast cancers. Moreover, we identified activation of the Pi3k/Akt/mTOR pathway in most tumors via mutations in Pten, Erbb2, Kras, and/or a recurrent Pip5k1c mutation that stabilizes the Pip5k1c protein and activates Pi3k/Akt/mTOR signaling. Another PIP5K1C family member, PIP5K1A, is coamplified with PI4KB in 18% of human breast cancer patients; both encode kinases that are responsible for production of the PI3K substrate, phosphatidylinositol 4,5-bisphosphate. Thus, the TP53R248W mutation and PI3K/AKT/mTOR signaling are major cooperative events driving breast-cancer development. Additionally, a combination of two US Food and Drug Administration (FDA)-approved drugs, tigecycline and metformin, which target oxidative phosphorylation downstream of PI3K signaling, inhibited tumor cell growth and may be repurposed for breast-cancer treatment. These findings advance our understanding of how mutant p53 drives breast-tumor development and pinpoint the importance of PI3K/AKT/mTOR signaling, expanding combination therapies for breast-cancer treatment.

Men1 maintains exocrine pancreas homeostasis in response to inflammation and oncogenic stress.

A more comprehensive understanding of the molecular mechanisms underlying pancreatic diseases, including pancreatitis and cancer, is essential to improve clinical management. MEN1 has established roles in epigenetic regulation and tumor suppression in the endocrine pancreas; however, intriguing recent data suggest MEN1 may also function in the exocrine pancreas. Using physiologically relevant genetic mouse models, we provide direct evidence that Men1 is essential for exocrine pancreas homeostasis in response to inflammation and oncogenic stress. Men1 loss causes increased injury and impaired regeneration following acute caerulein-induced pancreatitis, leading to more severe damage, loss of the normal acinar compartment, and increased cytokeratin 19-positive metaplasias and immune cell infiltration. We further demonstrate the Men1 protein is stabilized in response to insult, and loss of Men1 is associated with the overexpression of proinflammatory Jund target genes, suggesting that loss of Men1-mediated repression of Jund activity is, at least in part, responsible for the impaired response. Finally, we demonstrate that Men1 loss significantly accelerates mutant Kras-dependent oncogenesis. Combined, this work establishes Men1 as an important mediator of pancreas homeostasis in vivo.

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.

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.

p53R245W Mutation Fuels Cancer Initiation and Metastases in NASH-driven Liver Tumorigenesis.

Obesity is a significant global health concern. Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH) are common risk factors for hepatocellular carcinoma (HCC) and are closely associated with metabolic comorbidities, including obesity and diabetes. The TP53 tumor suppressor is the most frequently mutated gene in liver cancers, with half of these alterations being missense mutations. These mutations produce highly abundant proteins in cancer cells which have both inhibitory effects on wildtype (WT) p53, and gain-of-function (GOF) activities that contribute to tumor progression. A Western diet increases p53 activity in the liver. To elucidate the functional consequences of Trp53 mutations in a NASH-driven liver tumorigenesis model, we generated somatic mouse models with Trp53 deletion or the missense hotspot mutant p53R245W only in hepatocytes and placed mice on a high-fat, choline-deficient diet. p53R245W in the presence of diet increased fatty liver, compensatory proliferation in the liver parenchyma, and enriched genes of tumor-promoting pathways such as KRAS signaling, MYC, and epithelial-mesenchymal transition when compared with controls in the premalignant liver. Moreover, p53R245W suppressed transcriptional activity of WT p53 in the liver in vivo under metabolic challenges, and shortened survival and doubling of HCC incidence as compared with control heterozygous mice. Complete loss of Trp53 also significantly accelerated liver tumor incidence and lowered time-to-tumor development compared with WT controls. p53R245W GOF properties increased carcinoma initiation, fueled mixed hepatocholangial carcinoma incidence, and tripled metastatic disease. Collectively, our in vivo studies indicate that p53R245W has stronger tumor promoting activities than Trp53 loss in the context of NASH. SIGNIFICANCE: Using somatic NASH-driven mouse models with p53 deletion or mutant p53R245W only in hepatocytes, we discovered that p53R245W increased carcinoma initiation, fueled hepatocholangial carcinoma incidence, and tripled metastases.

Context matters - Daxx and Atrx are not robust tumor suppressors in the murine endocrine pancreas.

Genome sequencing has revealed the importance of epigenetic regulators in tumorigenesis. The genes encoding the chromatin remodeling complex DAXX:ATRX are frequently mutated in pancreatic neuroendocrine tumors; however, the underlying mechanisms of how mutations contribute to tumorigenesis are only partially understood, in part because of the lack of relevant preclinical models. Here, we used genetically engineered mouse models combined with environmental stress to evaluate the tumor suppressor functions of Daxx and Atrx in the mouse pancreas. Daxx or Atrx loss, alone or in combination with Men1 loss, did not drive or accelerate pancreatic neuroendocrine tumorigenesis. Moreover, Daxx loss did not cooperate with environmental stresses (ionizing radiation or pancreatitis) or with the loss of other tumor suppressors (Pten or p53) to promote pancreatic neuroendocrine tumorigenesis. However, owing to promiscuity of the Cre promoter used, hepatocellular carcinomas and osteosarcomas were observed in some instances. Overall, our findings suggest that Daxx and Atrx are not robust tumor suppressors in the endocrine pancreas of mice and indicate that the context of a human genome is essential for tumorigenesis. This article has an associated First Person interview with the first author of the paper.

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.

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.

Daxx maintains endogenous retroviral silencing and restricts cellular plasticity in vivo.

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.

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.

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.

High levels of the p53 inhibitor MDM4 in head and neck squamous carcinomas.

The p53 tumor suppressor is mutated in most 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. In addition, 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 3 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.

Therapeutic efficacy of p53 restoration in Mdm2-overexpressing tumors.

UNLABELLED: 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, for example, upregulation 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. IMPLICATIONS: Restoration of wild-type p53 expression in Mdm2-overexpressing tumors suppresses tumor growth, which represents a potential clinical strategy to treat tumors with high levels of Mdm2.