Activation of the Snail transcription factor induces Mdm2 gene expression.

Epithelial-like tumor cells can become metastatic by undergoing molecular and phenotypic reprogramming in a process referred to as epithelial-to-mesenchymal transition (EMT). In response to EMT genes that promote migration and condition the tumor microenvironment to permit intravasation into the bloodstream, dissemination and extravasation into new organs are induced. While the mutant p53 has been implicated in extravasation, one negative regulator of p53, the oncogene murine double minute-2 gene (Mdm2), is required in the early stages of metastasis and the driver of EMT. This activity is independent of Mdm2's role in the p53-Mdm2 autoregulatory feedback loop. Herein, we examine the EMT transcription factor Snail as a downstream effector of kinase signaling pathways. We show that the activation of upstream receptors and KRas signaling increase Snail levels. Snail binds to Ebox DNA motifs, and Mdm2 has two Ebox DNA-binding domains in the second promoter. Snail binds to the second Ebox and induces Mdm2 gene expression. Knockdown of endogenous Snail by shRNA shows a decrease in Mdm2 and is associated with reduced migration. The reintroduction of Mdm2 in shSnail cells restores cellular migration. These data integrate upstream pathways that induce Snail-Mdm2 to promote the metastasis of tumor cells.

The Effect of Replication Protein A Inhibition and Post-Translational Modification on ATR Kinase Signaling.

The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair towards ensuring genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions, termed RPA-DBi, and RPA protein-protein interactions, termed RPA-PPIi; both activities are required for ATR activation. Here, we employ a biochemically reconstituted ATR kinase signaling pathway and demonstrate that both RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of downstream target proteins. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation has no effect on ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway targeted cancer therapeutics.

The effect of replication protein A inhibition and post-translational modification on ATR kinase signaling.

The ATR kinase responds to elevated levels of single-stranded DNA (ssDNA) to activate the G2/M checkpoint, regulate origin utilization, preserve fork stability, and allow DNA repair to ensure genome integrity. The intrinsic replication stress in cancer cells makes this pathway an attractive therapeutic target. The ssDNA that drives ATR signaling is sensed by the ssDNA-binding protein replication protein A (RPA), which acts as a platform for ATRIP recruitment and subsequent ATR activation by TopBP1. We have developed chemical RPA inhibitors (RPAi) that block RPA-ssDNA interactions (RPA-DBi) and RPA protein-protein interactions (RPA-PPIi); both activities are required for ATR activation. Here, we biochemically reconstitute the ATR kinase signaling pathway and demonstrate that RPA-DBi and RPA-PPIi abrogate ATR-dependent phosphorylation of target proteins with selectivity advantages over active site ATR inhibitors. We demonstrate that RPA post-translational modifications (PTMs) impact ATR kinase activation but do not alter sensitivity to RPAi. Specifically, phosphorylation of RPA32 and TopBP1 stimulate, while RPA70 acetylation does not affect ATR phosphorylation of target proteins. Collectively, this work reveals the RPAi mechanism of action to inhibit ATR signaling that can be regulated by RPA PTMs and offers insight into the anti-cancer activity of ATR pathway-targeted cancer therapeutics.

Induction of the Mdm2 gene and protein by kinase signaling pathways is repressed by the pVHL tumor suppressor.

The tumor suppressor von Hippel-Lindau, pVHL, is a multifaceted protein. One function is to dock to the hypoxia-inducible transcription factor (HIF) and recruit a larger protein complex that destabilizes HIF via ubiquitination, preventing angiogenesis and tumor development. pVHL also binds to the tumor suppressor p53 to activate specific p53 target genes. The oncogene Mdm2 impairs the formation of the p53-pVHL complex and activation of downstream genes by conjugating nedd8 to pVHL. While Mdm2 can impact p53 and pVHL, how pVHL may impact Mdm2 is unclear. Like p53 somatic mutations, point mutations are evident in pVHL that are common in renal clear cell carcinomas (RCC). In patients with RCC, Mdm2 levels are elevated, and we examined whether there was a relationship between Mdm2 and pVHL. TCGA and DepMap analysis revealed that mdm2 gene expression was elevated in RCC with vhl point mutations or copy number loss. In pVHL reconstituted or deleted isogenetically match RCC or MEF cell lines, Mdm2 was decreased in the presence of pVHL. Furthermore, through analysis using genetic and pharmacological approaches, we show that pVHL represses Mdm2 gene expression by blocking the MAPK-Ets signaling pathway and blocks Akt-mediated phosphorylation and stabilization of Mdm2. Mdm2 inhibition results in an increase in the p53-p21 pathway to impede cell growth. This finding shows how pVHL can indirectly impact the function of Mdm2 by regulating signaling pathways to restrict cell growth.

Alteration of tumor suppressors changes the endometrial tumor spectrum.

The most common gynecological cancer in Europe and the United States is endometrial. Like most cancers, early-stage endometrial cancer has a more favorable prognosis, while high-grade, including endometrioid and nonendometrioid, has the worst prognosis. In endometrioid human tumors, the tumor suppressor genes PTEN and p53 (Trp53) are frequently altered or lost, as identified in datasets from The Cancer Genome Atlas. These suppressors' somatic mutations or loss of gene expression can lead to neoplastic development, tumor progression, and therapeutic resistance. In addition, somatic missense mutations are prevalent in another tumor suppressor, the F-box and WD repeats containing 7 (FBXW7). FBXW7 is part of the SCF-ßTrCP ubiquitin complex that signals protein destruction. Specifically, FBXW7 is responsible for binding and facilitating the destabilization of proteins involved in proliferation and migration. Losing the function of multiple tumor suppressors could activate pathways involved in neoplastic progression, malignancy, therapeutic resistance, and formation of different tumor subtypes. The study by Brown et al in this issue of EMBO Mol Med (Brown et al, 2023) provides insight into the complexity of tumor suppressor mutations in malignant endometrial cancer.

Author Correction: Mutant p53 drives clonal hematopoiesis through modulating epigenetic pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mdm2-mediated neddylation of pVHL blocks the induction of antiangiogenic factors.

Mutations in the tumor suppressor TP53 are rare in renal cell carcinomas. p53 is a key factor for inducing antiangiogenic genes and RCC are highly vascularized, which suggests that p53 is inactive in these tumors. One regulator of p53 is the Mdm2 oncogene, which is correlated with high-grade, metastatic tumors. However, the sole activity of Mdm2 is not just to regulate p53, but it can also function independent of p53 to regulate the early stages of metastasis. Here, we report that the oncoprotein Mdm2 can bind directly to the tumor suppressor VHL, and conjugate nedd8 to VHL within a region that is important for the p53-VHL interaction. Nedd8 conjugated VHL is unable to bind to p53 thereby preventing the induction of antiangiogenic factors. These results highlight a previously unknown oncogenic function of Mdm2 during the progression of cancer to promote angiogenesis through the regulation of VHL. Thus, the Mdm2-VHL interaction represents a pathway that impacts tumor angiogenesis.

Mutant p53 drives clonal hematopoiesis through modulating epigenetic pathway.

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. Here we discover that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Furthermore, genetic and pharmacological inhibition of EZH2 decreases the repopulating potential of p53 mutant HSPCs. Thus, we uncover an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.

Protein phosphatase 5 and the tumor suppressor p53 down-regulate each other's activities in mice.

Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53+/-pp5+/- or p53+/-pp5-/- mice revealed that complete loss of PP5 reduces tumorigenesis in the p53+/- mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.

The proto-oncogene function of Mdm2 in bone.

Mouse double minute 2 (Mdm2) is a multifaceted oncoprotein that is highly regulated with distinct domains capable of cellular transformation. Loss of Mdm2 is embryonically lethal, making it difficult to study in a mouse model without additional genetic alterations. Global overexpression through increased Mdm2 gene copy number (Mdm2Tg ) results in the development of hematopoietic neoplasms and sarcomas in adult animals. In these mice, we found an increase in osteoblastogenesis, differentiation, and a high bone mass phenotype. Since it was difficult to discern the cell lineage that generated this phenotype, we generated osteoblast-specific Mdm2 overexpressing (Mdm2TgOb ) mice in 2 different strains, C57BL/6 and DBA. These mice did not develop malignancies; however, these animals and the MG63 human osteosarcoma cell line with high levels of Mdm2 showed an increase in bone mineralization. Importantly, overexpression of Mdm2 corrected age-related bone loss in mice, providing a role for the proto-oncogenic activity of Mdm2 in bone health of adult animals.

Nuclear PTEN enhances the maturation of a microRNA regulon to limit MyD88-dependent susceptibility to sepsis.

Sepsis-induced organ damage is caused by systemic inflammatory response syndrome (SIRS), which results in substantial comorbidities. Therefore, it is of medical importance to identify molecular brakes that can be exploited to dampen inflammation and prevent the development of SIRS. We investigated the role of phosphatase and tensin homolog (PTEN) in suppressing SIRS, increasing microbial clearance, and preventing lung damage. Septic patients and mice with sepsis exhibited increased PTEN expression in leukocytes. Myeloid-specific Pten deletion in an animal model of sepsis increased bacterial loads and cytokine production, which depended on enhanced myeloid differentiation primary response gene 88 (MyD88) abundance and resulted in mortality. PTEN-mediated induction of the microRNAs (miRNAs) miR125b and miR203b reduced the abundance of MyD88. Loss- and gain-of-function assays demonstrated that PTEN induced miRNA production by associating with and facilitating the nuclear localization of Drosha-Dgcr8, part of the miRNA-processing complex. Reconstitution of PTEN-deficient mouse embryonic fibroblasts with a mutant form of PTEN that does not localize to the nucleus resulted in retention of Drosha-Dgcr8 in the cytoplasm and impaired production of mature miRNAs. Thus, we identified a regulatory pathway involving nuclear PTEN-mediated miRNA generation that limits the production of MyD88 and thereby limits sepsis-associated mortality.

Mutant and wild-type p53 form complexes with p73 upon phosphorylation by the kinase JNK.

The transcription factors p53 and p73 are critical to the induction of apoptotic cell death, particularly in response to cell stress that activates c-Jun N-terminal kinase (JNK). Mutations in the DNA-binding domain of p53, which are commonly seen in cancers, result in conformational changes that enable p53 to interact with and inhibit p73, thereby suppressing apoptosis. In contrast, wild-type p53 reportedly does not interact with p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53, as well as mutant p53, to form a complex with p73. Structural algorithms predicted that phosphorylation of Thr81 exposes the DNA-binding domain in p53 to enable its binding to p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes [such as those encoding p53-up-regulated modulator of apoptosis (PUMA) and Bcl-2-associated X protein (BAX)] and, subsequently, the induction of apoptosis in response to JNK activation by cell stress in various cells. Thus, JNK phosphorylation of mutant and wild-type p53 promotes the formation of a p53/p73 complex that determines cell fate: apoptosis in the context of wild-type p53 or cell survival in the context of the mutant. These findings refine our current understanding of both the mechanistic links between p53 and p73 and the functional role for Thr81 phosphorylation.

The fate of murine double minute X (MdmX) is dictated by distinct signaling pathways through murine double minute 2 (Mdm2).

Mouse double minute 2 (Mdm2) and MdmX dimerize in response to low levels of genotoxic stress to function in a ubiquitinating complex, which signals for destabilization of p53. Under growth conditions, Mdm2 functions as a neddylating ligase, but the importance and extent of MdmX involvement in this process are largely unknown. Here we show that when Mdm2 functions as a neddylating enzyme, MdmX is stabilized. Furthermore, we demonstrate that under growth conditions, MdmX enhances the neddylation activity of Mdm2 on p53 and is a substrate for neddylation itself. Importantly, MdmX knockdown in MCF-7 breast cancer cells resulted in diminished neddylated p53, suggesting that MdmX is important for Mdm2-mediated neddylation. Supporting this finding, the lack of MdmX in transient assays or in p53/MdmX-/- MEFs results in decreased or altered neddylation of p53 respectively; therefore, MdmX is a critical component of the Mdm2-mediated neddylating complex. c-Src is the upstream activator of this Mdm2-MdmX neddylating pathway and loss of Src signaling leads to the destabilization of MdmX that is dependent on the RING (Really Interesting New Gene) domain of MdmX. Treatment with a small molecule inhibitor of neddylation, MLN4924, results in the activation of Ataxia Telangiectasia Mutated (ATM). ATM phosphorylates Mdm2, converting Mdm2 to a ubiquitinating enzyme which leads to the destabilization of MdmX. These data show how distinct signaling pathways engage neddylating or ubiquitinating activities and impact the Mdm2-MdmX axis.

Early-Stage Metastasis Requires Mdm2 and Not p53 Gain of Function.

Metastasis of cancer cells to distant organ systems is a complex process that is initiated with the programming of cells in the primary tumor. The formation of distant metastatic foci is correlated with poor prognosis and limited effective treatment options. We and others have correlated Mouse double minute 2 (Mdm2) with metastasis; however, the mechanisms involved have not been elucidated. Here, it is reported that shRNA-mediated silencing of Mdm2 inhibits epithelial-mesenchymal transition (EMT) and cell migration. In vivo analysis demonstrates that silencing Mdm2 in both post-EMT and basal/triple-negative breast cancers resulted in decreased primary tumor vasculature, circulating tumor cells, and metastatic lung foci. Combined, these results demonstrate the importance of Mdm2 in orchestrating the initial stages of migration and metastasis.Implication: Mdm2 is the major factor in the initiation of metastasis. Mol Cancer Res; 15(11); 1598-607. ©2017 AACR.

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist.

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

Emerging Non-Canonical Functions and Regulation by p53: p53 and Stemness.

Since its discovery nearly 40 years ago, p53 has ascended to the forefront of investigated genes and proteins across diverse research disciplines and is recognized most exclusively for its role in cancer as a tumor suppressor. Levine and Oren (2009) reviewed the evolution of p53 detailing the significant discoveries of each decade since its first report in 1979. In this review, we will highlight the emerging non-canonical functions and regulation of p53 in stem cells. We will focus on general themes shared among p53's functions in non-malignant stem cells and cancer stem-like cells (CSCs) and the influence of p53 on the microenvironment and CSC niche. We will also examine p53 gain of function (GOF) roles in stemness. Mutant p53 (mutp53) GOFs that lead to survival, drug resistance and colonization are reviewed in the context of the acquisition of advantageous transformation processes, such as differentiation and dedifferentiation, epithelial-to-mesenchymal transition (EMT) and stem cell senescence and quiescence. Finally, we will conclude with therapeutic strategies that restore wild-type p53 (wtp53) function in cancer and CSCs, including RING finger E3 ligases and CSC maintenance. The mechanisms by which wtp53 and mutp53 influence stemness in non-malignant stem cells and CSCs or tumor-initiating cells (TICs) are poorly understood thus far. Further elucidation of p53's effects on stemness could lead to novel therapeutic strategies in cancer research.

Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model.

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.