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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

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

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 Trp53 R245W mouse model (equivalent to the TP53 R248W 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 TP53 R248W 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.

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.

Table S1 shows predicted off target sites for p53TD mutant mice. Table S2 shows upregulated PPAR target genes in p53AD vs p53-null. Table S3 shows ATAC-seq and RNA-seq congruent genes in p53AD vs p53 null. Table S4 shows p53half site analysis in PPAR target genes Table S5 shows control sequences used for p53 half site analysis Table S6 shows altered genes in p53AD-derived thymic lymphomas Table S7 shows primers used for genotyping of p53 mutant mice Table S8 shows primers used for qRT-PCR Table S9 shows antibodies used for flow cytometry Figure S1 shows extended data for generation of knock-in mouse models and features of p53TD mutant proteins and mice. Figure S2 shows homozygous mutant mice survival curves as well as tumor data. Figure S3 shows exome sequencing results for thymic lymphomas and hemangiosarcomas derived from p53 monomer and dimer mutant mice. Figure S4 shows characterization of stress responses of p53TD mutants. Figure S5 shows RNA-sequencing results for p53AD and p53RP, and Mdm2 genetic deletion rescue experiments. Figure S6 shows RNA-seq results of basal p53AD vs p53-null thymi, upstream regulator analysis, and ATAC-seq results for specific loci. Figure S7 shows extended data for p53 dimer mutant novel activities (co-IP, motif analysis, other dimer mutants activities, and tumor data) Figure S8 shows ferroptosis and scRNA-seq extended data Figure S9 shows diagram summarizing findings within this study.

Representative pictures of the gross morphology of Mdm4Δ2/Δ2 p53+�- embryos.

p73 loss does not rescue the Mdm4-deficiency brain phenotype.

Supplementary Figure 3

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.Visual Overview: http://mcr.aacrjournals.org/content/12/6/901/F1.large.jpg.Mol Cancer Res; 12(6); 901–11. ©2014 AACR.

PDF file - 354K, Figure S1. Tumor-free survival curves of the Mdm2Tg p53Neo/Neo and Mdm2Tg p53Neo/Neo CreER mice. Figure S2. Analysis of p53Neo recombination in tamoxifen-treated Mdm2Tg p53Neo/Neo CreER tumors. Figure S3. Initial tumor volumes of the Mdm2Tg p53Neo/Neo and Mdm2Tg p53Neo/Neo CreER angiosarcomas. Figure S4. p53 target gene p21 expression in tamoxifen-treated spontaneous Mdm2Tg p53Neo/Neo and Mdm2Tg p53Neo/Neo CreER angiosarcomas. Figure S5. PCR analysis of p53Neo allele recombination upon tamoxifen treatment in transplanted Mdm2Tg p53Neo/Neo CreER angiosarcomas. Figure S6. Effects of tamoxifen on the Mdm2Tgp53Neo/Neo angiosarcomas. Table S1. List of Mdm2Tg p53Neo/Neo and Mdm2Tg p53Neo/Neo CreER angiosarcomas monitored in the MRI study.

Mutant p53-associated genes

Supplementary Data from 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.

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, Mdm4Tg1 and Mdm4Tg15, 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 Mdm4Tg1 p53+/− mice. These mice had significantly accelerated tumorigenesis and a distinct tumor spectrum with more carcinomas and significantly fewer lymphomas than p53+/− or Mdm4Tg1 mice. Importantly, the remaining wild-type p53 allele was retained in most Mdm4Tg1 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. Cancer Res; 70(18); 7148–54. ©2010 AACR.

Supplementary Figure 3 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Supplementary Figure 1 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Supplementary Methods, Figure Legends 1-3 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Supplementary Table 1 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Supplementary Figure 2 from Spontaneous Tumorigenesis in Mice Overexpressing the p53-Negative Regulator Mdm4

Whether p53, either wild type (WT) or mutant, plays cell-specific or uniform role remains controversial. Using The Cancer Genome Atlas, we examined p53 in the lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), two lung cancers with different cellular origins and frequent

The developmental origins of mesenchymal progenitor cells (MPCs) and molecular machineries regulating their fate and differentiation are far from defined owing to their complexity. Osteoblasts and adipocytes are descended from common MPCs. Their fates are collectively determined by an orchestra of pathways in response to physiological and external cues. The canonical Wnt pathway signals MPCs to commit to osteogenic differentiation at the expense of adipogenic fate. In contrast to ß-catenin, p53’s anti-osteogenic function is much less understood. Both activities are thought to be achieved through targeting Runx2 and/or Osterix (Osx, Sp7) transcription. Precisely, how Osx activity is dictated by ß-catenin or p53 is not clarified and represents a knowledge gap that, until now, has largely been taken for granted. Using conditional lineage-tracing mice, we demonstrated that chondrocytes gave rise to a sizable fraction of MPCs, which served as progenitors of chondrocyte-derived osteoblasts (Chon-ob). Wnt/ß-catenin activity was only required at the stage of chondrocyte-derived mesenchymal progenitor (C-MPC) to Chon-ob differentiation. ß-catenin– C-MPCs lost osteogenic ability and favored adipogenesis. Mechanistically, we discovered that p53 activity was elevated in ß-catenin– MPCs including ß-catenin– C-MPCs and deleting p53 from the ß-catenin– MPCs fully restored osteogenesis. While high levels of p53 were present in the nuclei of ß-catenin– MPCs, Osx was confined to the cytoplasm, implying a mechanism that did not involve direct p53-Osx interaction. Furthermore, we found that p53’s anti-osteogenic activity was dependent on its DNA-binding ability. Our findings identify chondrocytes as an additional source for MPCs and indicate that Wnt/ß-catenin discretely regulates chondrocyte to C-MPC and the subsequent C-MPC to osteoblast developments. Most of all we unveil a previously unrecognized functional link between ß-catenin and p53, placing p53’s negative role in the context of Wnt/ß-catenin signaling-induced MPC osteogenic differentiation.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor FOXA1, activating its transcriptional network while promoting WNT/β-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and β-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis. Significance: Oncogenic KRAS and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic KRAS effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis. This article is highlighted in the In This Issue feature, p. 1861

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.

The majority of p53 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 in vivo, we generated a novel germline p53R245W allele (contact mutation) and compared it to existing p53R172H (structural mutation) and p53R270H (contact mutation) alleles. Thymocytes from heterozygous mice showed all three hot-spot 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 p53R245W/+ and p53R270H/+ mice, but not p53R172H/+ mice, was significantly shorter than that of p53+/- mice, demonstrating p53 mutation-specific GOF contributions to tumor development. Furthermore, p53R245W/+ and p53R270H/+ mice had more osteosarcoma metastases than p53R172H/+ mice, indicating that these two contact mutants have stronger GOF in driving osteosarcoma metastasis. Omics analyses using RNA-sequencing data from p53R172H/+, p53R245W/+, and p53R270H/+ primary osteosarcomas in comparison to p53+/- indicated GOF was mediated by distinct pathways among the three mutations. Thus, both the inhibitory effect of mutant over WT p53, and GOF activities of mutant p53 contributed to tumorigenesis in vivo. Targeting p53 mutant-specific pathways may be important for therapeutic outcomes in osteosarcomas. Citation Format: Shunbin Xiong, Dhruv Chachad, Yun Zhang, Jovanka Gencel-Augusto, Mario Sirito, Vinod Pant, Peirong Yang, Chang Sun, Guillermina Lozano. Differences in gain-of-function and inhibitory effects amongst p53 mutants in vivo [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A042.

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 Mdm2(P2/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.

BACKGROUND The objective of the present research was to explore the prevalence, risk, and prognostic factors associated with bone metastases (BM) in newly diagnosed hepatocellular carcinoma (HCC) patients. MATERIAL AND METHODS From 36 507 HCC patients who were registered in Surveillance, Epidemiology, and End Results (SEER) database, we enrolled 1263 with BM at the initial diagnosis of HCC from 2010 to 2014. Kaplan-Meier curves and log-rank tests were used to estimate overall survival for different subgroups. Univariate and multivariate logistic and Cox regression analyses were performed to identify risk factors and independent prognostic factors for BM. RESULTS A total of 1567 (4.29%) HCC patients were detected with BM at initial diagnosis. Male sex, unmarried status, higher T stage, lymph node involvement, intrahepatic metastases, and extrahepatic metastases (lung or brain) were positively associated with BM. The median survival of the patients was 3.00 months (95% CI: 2.77-3.24 months). Marital status and primary tumor surgery were independently associated with the better survival. CONCLUSIONS A list of factors associated with BM occurrence and the prognosis of the advanced HCC patients with BM were found. These associated factors may provide a reference for BM screening in HCC and guide prophylactic treatment in clinical settings.

MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/β-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of β-Catenin in a p53-independent manner. Wnt/β-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/β-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/β-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

p53 is one of the most intensively studied tumor suppressors. It transcriptionally regulates a broad range of genes to modulate a series of cellular events, including DNA damage repair, cell cycle arrest, senescence, apoptosis, ferroptosis, autophagy, and metabolic remodeling, which are fundamental for both development and cancer. This review discusses the role of p53 in brain development, neural stem cell regulation and the mechanisms of inactivating p53 in gliomas. p53 null or p53 mutant mice show female biased exencephaly, potentially due to X chromosome inactivation failure and/or hormone-related gene expression. Oxidative cellular status, increased PI3K/Akt signaling, elevated ID1, and metabolism are all implicated in p53-loss induced neurogenesis. However, p53 has also been shown to promote neuronal differentiation. In addition, p53 mutations are frequently identified in brain tumors, especially glioblastomas. Mechanisms underlying p53 inactivation in brain tumor cells include disruption of p53 protein stability, gene expression and transactivation potential as well as p53 gene loss or mutation. Loss of p53 function and gain-of-function of mutant p53 are both implicated in brain development and tumor genesis. Further understanding of the role of p53 in the brain may provide therapeutic insights for brain developmental syndromes and cancer.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate cyclic AMP responsive element binding protein 1 (CREB1) to allow physical interactions with mutant p53 that hyperactivate multiple pro-metastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the pro-metastatic, pioneer transcription factor, FOXA1, activating its transcriptional network while promoting WNT/β-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and β-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis.

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., Mutations in TP53 gene are very common in cancer development. Here the authors take advantage of murine models to show that somatic Trp53 mutations differentially drive breast cancer and evolution of metastases.

Background/Aims: Gain-of-function of mutant p53 is associated with a high rate of lung metastasisinosteosarcoma. To investigate the mechanism of mutant p53-induced osteosarcoma metastasis, expression array analysis was performed, comparing non-metastatic osteosarcomas from p53(+/-) mice with metastatic osteosarcomas from p53(R172H/+) mice. Onzin (Plac8) was identified as one of the genes upregulated in p 53(R172H/+) mouse metastatic osteosarcomas. Accordingly, we investigated the role of ONZIN in human osteosarcoma metastasis. Methods: ONZIN function and its downstream targets were examined in osteosarcoma cell lines. Assays related to tumorigenesis and metastasis, including cell migration, invasion, clonogenic survival, and soft agar colony formation, were performed in osteosarcoma cells. Additionally, mouse xenograft models were used to examine the role of ONZIN overpression in tumorigenesis in vivo. Lastly, 87 osteosarcoma patients were recruited to investigate the clinical relevance of ONZIN overexpression in metastasis and prognosis. Results: ONZIN overexpression enhanced osteosarcoma cell proliferation, clonogenic survival, migration, and invasion independent of p53 status. Furthermore, ONZIN overexpression induced CXCL5 upregulation and resulted in increased ERK phosphorylation, which contributed to more aggressive osteosarcoma metastatic phenotypes. More importantly, overexpression of ONZIN in human osteosarcoma patients was closely associated with lung metastasis, poor prognoses, and survival. Conclusions: Overexpression of ONZIN promotes osteosarcoma progression and metastasis, and can serve as a clinical biomarker for osteosarcoma metastasis and prognosis. (C) 2018 The Author(s) Published by S. Karger AG, Basel

MDM2, an E3 ubiquitin ligase, is a potent inhibitor of the p53 tumor suppressor and is elevated in many human cancers that retain wild-type p53. MDM2 SNP309G is a functional polymorphism that results in elevated levels of MDM2 (due to enhanced SP1 binding to the MDM2 promoter) thus decreasing p53 activity. Mdm2SNP309G/G mice are more prone to spontaneous tumor formation than Mdm2SNP309T/T mice, providing direct evidence for the impact of this SNP in tumor development. We asked whether environmental factors impact SNP309G function and show that SNP309G cooperates with ionizing radiation to exacerbate tumor development. Surprisingly, ultraviolet B light or Benzo(a)pyrene exposure of skin shows that SNP309G allele actually protects against squamous cell carcinoma susceptibility. These contrasting differences led us to interrogate the mechanism by which Mdm2 SNP309 regulates tumor susceptibility in a tissue-specific manner. Although basal Mdm2 levels were significantly higher in most tissues in Mdm2SNP309G/G mice compared with Mdm2SNP309T/T mice, they were significantly lower in Mdm2SNP309G/G keratinocytes, the cell-type susceptible to squamous cell carcinoma. The assessment of potential transcriptional regulators in ENCODE ChIP-seq database identified transcriptional repressor E2F6 as a possible negative regulator of MDM2 expression. Our data show that E2F6 suppresses Mdm2 expression in cells harboring the SNP309G allele but not the SNP309T allele. Thus, Mdm2 SNP309G exhibits tissue-specific regulation and differentially impacts cancer risk.

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 MDMX is overexpressed in the vast majority of patients with AML; however, a causative role of MDMX in leukemogenesis has not yet been investigated. Mdmx transgenic mice (Mdmx-Tg) displayed increased number, proliferation and competitive advantage of hematopoietic stem cells compared to WT littermates. Crossing Mdmx-Tg with preleukemic mouse models representing frequent alterations in human AML, we generated three distinct compound models. We found that Mdmx overexpression triggered progression of each of the different chronic/asymptomatic preleukemic conditions to overt AML, and thereby markedly shortened survival. RNA sequencing revealed that Mdmx overexpression exerted this function through activation of Wnt/b-Catenin signaling in pre-LSC. Mechanistically, we found that MDMX bound CK1a, a canonical degrader of b-Catenin, and led to accumulation and nuclear translocation of b-Catenin. WNT/b-Catenin inhibitors or exogenous overexpression of CK1a were both effective in reversing Mdmx-induced pre-LSC properties, and showed further synergism in combination with MDMX inhibitor treatment. Competitive advantage and accumulation of b-Catenin upon Mdmx overexpression were still seen upon crossing into a p53-/- background, demonstrating that Mdmx-driven WNT/b-Catenin activation and its functional consequences are, at least part, independent of p53. Lastly, upregulation of WNT/b-Catenin pathways correlated with MDMX overexpression in large cohorts of patients with MDS and was associated with increased risk of progression to AML and poor survival. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML switch mechanism effective in different genetically-driven disease subtypes, and reveals WNT/b-Catenin as a novel, non-canonical p53-independent MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most p53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis. However, the extent of direct cooperation or synergism between KRAS effectors and mutant p53 remain undefined. To study how KRAS effectors and mutant p53 cooperate to drive PDAC development, we developed a novel somatic model that expresses mutant KRAS and p53 only in pancreas tumor cells, leaving the tumor microenvironment and immune system intact. PDAC genetically engineered mouse models (GEMMs) were generated by crossing a conditional oncogenic KRasG12D (K) allele with a conditional null p53 allele (Pfl) or a novel, conditional knock-in mutant p53R172H allele (Pwm) that selectively expresses mutant p53 in tumor cells while preserving wildtype p53 expression in all other animal cells. The incidence of metastatic lesions in the liver and lungs of KPwmC mice was over 2-fold greater (p=.03) relative to KPflC mice. Through orthogonal analyses of this model and human PDAC samples, we show that oncogenic KRAS effectors phosphorylate and activate cyclic AMP responsive element binding protein 1 (CREB1), allowing physical interactions with mutant p53 to transcriptionally upregulate the pro-metastatic transcription factor, FOXA1, which enhances β-catenin stabilization and activity. Targeting KRAS and mutant p53 cooperativity through pharmacologic inhibition of CREB1 dramatically dampens FOXA1 expression and PDAC metastasis. Our findings demonstrate a direct, mechanistic link between key oncogenic KRAS signaling elements and mutant p53 that result in a broad, multiplexed activation of cancer-associated transcriptional networks. Moreover, we identify CREB1 as a viable therapeutic strategy to undermine oncogenic KRAS and mutant p53 cooperation to mitigate PDAC metastasis. Citation Format: Michael Paul Kim, Xinqun Li, Jenying Deng, Yun Zhang, Bingbing Dai, Kendra Allton, Tara Hughes, Christian Siangco, Jithesh Augustine, Yaan Kang, Joy M. McDaniel, Shunbin Xiong, Eugene Koay, Florencia McAllister, Christopher Bristow, Timothy Heffernan, Anirban Maitra, Bin Liu, Michelle Barton, Amanda Wasylischen, Jason Fleming, Guillermina lozano. Mutant p53 and oncogenic KRAS converge on CREB1 to drive pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2417.

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.

Mdm2, the principal negative regulator of p53, is critical for survival, a fact clearly demonstrated by the p53-dependent death of germline or conditional mice following deletion of Mdm2. On the other hand, Mdm2 hypomorphic (Mdm2Puro/Δ7 − 12) or heterozygous (Mdm2+/−) mice that express either 30 or 50% of normal Mdm2 levels, respectively, are viable but present distinct phenotypes because of increased p53 activity. Mdm2 levels are also transcriptionally regulated by p53. We evaluated the significance of this reciprocal relationship in a new hypomorphic mouse model inheriting an aberrant Mdm2 allele with insertion of the neomycin cassette and deletion of 184-bp sequence in intron 3. These mice also carry mutations in the Mdm2 P2-promoter and thus express suboptimal levels of Mdm2 entirely encoded from the P1-promoter. Resulting mice exhibit abnormalities in skin pigmentation and reproductive tissue architecture, and are subfertile. Notably, all these phenotypes are rescued on a p53-null background. Furthermore, these phenotypes depend on distinct p53 downstream activities as genetic ablation of the pro-apoptotic gene Puma reverts the reproductive abnormalities but not skin hyperpigmentation, whereas deletion of cell cycle arrest gene p21 does not rescue either phenotype. Moreover, p53-mediated upregulation of Kitl influences skin pigmentation. Altogether, these data emphasize tissue-specific p53 activities that regulate cell fate.

The prognosis of metastatic osteosarcoma is dismal and a better understanding of the mechanisms underlying disease progression is essential to improve treatment options and patient outcomes. We previously demonstrated Pla2g16 overexpression in mouse osteosarcoma contributes to metastasis phenotypes and increased expression of PLA2G16 is associated with metastasis and poor prognosis in human tumors. To further examine the mechanisms through which PLA2G16 contributes to human osteosarcoma metastasis and explore the potential of PLA2G16 as a therapeutic target in osteosarcoma, we generated a panel of human osteosarcoma cell lines expressing different levels of PLA2G16. The functional analyses of these cell lines demonstrated high levels of PLA2G16 expression increased osteosarcoma cell migration, invasion, clonogenic survival, and anchorage-independent colony formation. Importantly, this activity was dependent on the phospholipase activity of PLA2G16. Additionally, PLA2G16 overexpression decreased the sensitivity of cells to a panel of chemotherapeutic agents. Analysis of downstream pathways revealed the pro-metastasis functions of PLA2G16 were mediated through the MAPK pathway, as knockdown of PLA2G16 decreased ERK1/2 phosphorylation and pharmacological inhibition of MEK significantly repressed PLA2G16 mediated cell migration and clonogenic survival. Furthermore, PLA2G16 overexpression promoted xenograft tumor growth in vivo, and these tumors exhibit increased ERK1/2 phosphorylation. Lastly, the expression of PLA2G16 is strongly correlated with the increased ERK1/2 phosphorylation in human osteosarcoma samples, and the combined lesions are associated with reduced overall and metastasis-free survival. Collectively, these results demonstrate increased PLA2G16 expression activates the MAPK pathway to enhance osteosarcoma metastasis and may be a novel therapeutic target for these cancers.

Table S3. Multivariable logistic regression for the presence of bone metastases at diagnosis of lung cancer. (PDF 228 kb)

Table S2. Univariate logistic regression for the presence of bone metastases at diagnosis of different subtypes of lung cancer. (PDF 298 kb)