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.

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.

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.

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.