P53 Transactivation Research Articles

Autoinhibition of MDMX by intramolecular p53 mimicry

The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions.

MDMX exerts its oncogenic activity via suppression of retinoblastoma protein.

Inactivation of the retinoblastoma protein (RB) has a major role in the development of human malignancies. We have previously shown that MDM2, an ubiquitin E3 ligase and major negative regulator of p53, binds to and promotes proteasome-mediated degradation of RB. MDMX, a homolog of MDM2, also binds to and inhibits p53 transactivation activity, yet it does not possess intrinsic ubiquitin ligase activity. Here, we show that MDMX binds to and promotes RB degradation in an MDM2-dependent manner. Specifically, the MDMX C-terminal ring domain binds to the RB C-pocket and enhances MDM2-RB interaction. Silencing MDMX induces RB accumulation, cell cycle arrest and senescence-like phenotypes, which are reverted by simultaneous RB knockdown. Furthermore, MDMX ablation leads to significant retardation of xenograft tumor growth, concomitant with RB accumulation. These results demonstrate that MDMX exerts oncogenic activity via suppression of RB, and suggest that both MDM2 and MDMX could be chemotherapeutic targets.

Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay.

Co-evolution of transcription factors (TFs) with their respective cis-regulatory network enhances functional diversity in the course of evolution. We present a new approach to investigate transactivation capacity of sequence-specific TFs in evolutionary studies. Saccharomyces cerevisiae was used as an in vivo test tube and p53 proteins derived from human and five commonly used animal models were chosen as proof of concept. p53 is a highly conserved master regulator of environmental stress responses. Previous reports indicated conserved p53 DNA binding specificity in vitro, even for evolutionary distant species. We used isogenic yeast strains where p53-dependent transactivation was measured towards chromosomally integrated p53 response elements (REs). Ten REs were chosen to sample a wide range of DNA binding affinity and transactivation capacity for human p53 and proteins were expressed at two levels using an inducible expression system. We showed that the assay is amenable to study thermo-sensitivity of frog p53, and that chimeric constructs containing an ectopic transactivation domain could be rapidly developed to enhance the activity of proteins, such as fruit fly p53, that are poorly effective in engaging the yeast transcriptional machinery. Changes in the profile of relative transactivation towards the ten REs were measured for each p53 protein and compared to the profile obtained with human p53. These results, which are largely independent from relative p53 protein levels, revealed widespread evolutionary divergence of p53 transactivation specificity, even between human and mouse p53. Fruit fly and human p53 exhibited the largest discrimination among REs while zebrafish p53 was the least selective.

Cardiac transcription factor Nkx2.5 interacts with p53 and modulates its activity

Transcription factor Nkx2.5, essential for heart development, regulates cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted (GATA4 and dHAND) or ubiquitous (p300) transcription regulators. Here we demonstrate that Nkx2.5 and p53 synergistically activate the promoter of the striated muscle stress responsive transcriptional cofactor Ankrd2, involved in coordination of proliferation and apoptosis during myogenic differentiation. Moreover, the p53 protein is able to interact with both wild type Nkx2.5 and its mutant ΔNkx2.5 (aa 1–198) found in patients with diverse cardiac malformations. Nkx2.5 interaction site of p53 maps to the C terminal region, while p53 binding site on Nkx2.5 lies outside its C terminus. In addition, overexpression of Nkx2.5 has a modulatory, promoter dependent effect on p53 transactivation, while the mutant significantly abolished p53 activity on the Mdm2, p21WAF1/CIP1 and Bax promoters. Their physical interaction contributes to the observed behavior in the case of the Mdm2 promoter. Our data provide a new evidence for the role of p53 in cardiac function through interaction with Nkx2.5.

Analysis of p53 transactivation domain mutants reveals Acad11 as a metabolic target important for p53 pro-survival function.

The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stresssignals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that inoncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.

Interactions of Chromatin Context, Binding Site Sequence Content, and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10−7) and correlated with stronger p53RE sequences (p<10−110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.

Transactivation of P53 by cypermethrin induced miR-200 and apoptosis in neuronal cells

Cypermethrin, a pyrethroid pesticide, has been shown to induce neurotoxicity in adult mammals. However, studies are also needed to explore its toxicity in developing brains and understand its mechanism of action in neurons.

Identification of the p53-Responsive Element in the Promoter Region of the Human Decorin Gene

We have obtained some evidence that shows that the decorin gene is the target of p53 transactivation. Luciferase reporter plasmid, which contained the promoter region between positions -252 and -205, was activated by p53 dose- dependently up to 170-fold. The promoter region involved a sequence, 5’-AGGCAAGTAG-3’, similar to p53-binding consensus sequence, 5’-PuPuPuC(A/T)(A/T)GPyPyPy-3’. Chromatin immunoprecipitation assay using p53 antibodies revealed that the region between -413 and -232 of the promoter of the decorin gene was co-precipitated with p53. p53- binding to this region was further demonstrated by electrophoretic mobility shift assay, in which the complex between decorin promoter DNA and proteins decreased by pretreatment with anti-p53 antibodies. The mRNA expression levels of decorin increased after treatment with p53-activating nutlin-3 greatly and with genotoxic reagent, adriamycin, to some extent. Consequently, decorin promoter is useful to evaluate the p53 transactivation ability.

Using Chemical Shifts to Generate Structural Ensembles for Intrinsically Disordered Proteins with Converged Distributions of Secondary Structure

A short segment of the disordered p53 transactivation domain (p53TAD) forms an amphipathic helix when bound to the E3 ubiquitin ligase, MDM2. In the unbound p53TAD, this short segment has transient helical secondary structure. Using a method that combines broad sampling of conformational space with re-weighting, it is shown that it is possible to generate multiple, independent structural ensembles that have highly similar secondary structure distributions for both p53TAD and a P27A mutant. Fractional amounts of transient helical secondary structure were found at the MDM2 binding site that are very similar to estimates based directly on experimental observations. Structures were identified in these ensembles containing segments that are highly similar to short p53 peptides bound to MDM2, even though the ensembles were re-weighted using unbound experimental data. Ensembles were generated using chemical shift data (alpha carbon only, or in combination with other chemical shifts) and cross-validated by predic...

High content screening biosensor assay to identify disruptors of p53-hDM2 protein-protein interactions.

This chapter describes the implementation of the p53-hDM2 protein-protein interaction (PPI) biosensor (PPIB) HCS assay to identify disruptors of p53-hDM2 PPIs. Recombinant adenovirus expression constructs were generated bearing the individual p53-GFP and hDM2-RFP PPI partners. The N-terminal p53 transactivating domain that contains the binding site for hDM2 is expressed as a GFP fusion protein that is targeted and anchored in the nucleolus of infected cells by a nuclear localization (NLS) sequence. The p53-GFP biosensor is localized to the nucleolus to enhance and facilitate the image acquisition and analysis of the PPIs. The N-terminus of hDM2 encodes the domain for binding to the transactivating domain of p53, and is expressed as a RFP fusion protein that includes both an NLS and a nuclear export sequence (NES). In U-2 OS cells co-infected with both adenovirus constructs, the binding interactions between hDM2 and p53 result in both biosensors becoming co-localized within the nucleolus. Upon disruption of the p53-hDM2 PPIs, the p53-GFP biosensor remains in the nucleolus while the shuttling hDM2-RFP biosensor redistributes into the cytoplasm. p53-hDM2 PPIs are measured by acquiring fluorescent images of cells co-infected with both adenovirus biosensors on an automated HCS imaging platform and using an image analysis algorithm to quantify the relative distribution of the hDM2-RFP shuttling component of the biosensor between the cytoplasm and nuclear regions of compound treated cells.

Using chemical shifts to generate structural ensembles for intrinsically disordered proteins with converged distributions of secondary structure.

A short segment of the disordered p53 transactivation domain (p53TAD) forms an amphipathic helix when bound to the E3 ubiquitin ligase, MDM2. In the unbound p53TAD, this short segment has transient helical secondary structure. Using a method that combines broad sampling of conformational space with re-weighting, it is shown that it is possible to generate multiple, independent structural ensembles that have highly similar secondary structure distributions for both p53TAD and a P27A mutant. Fractional amounts of transient helical secondary structure were found at the MDM2 binding site that are very similar to estimates based directly on experimental observations. Structures were identified in these ensembles containing segments that are highly similar to short p53 peptides bound to MDM2, even though the ensembles were re-weighted using unbound experimental data. Ensembles were generated using chemical shift data (alpha carbon only, or in combination with other chemical shifts) and cross-validated by predicting residual dipolar couplings. We think this ensemble generator could be used to predict the bound state structure of protein interaction sites in IDPs if there are detectable amounts of matching transient secondary structure in the unbound state.

Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.

Activation of p53 By Novel MDM2 Antagonist RG7388 Overcomes AML Inherent and Acquired Resistance to Bcl-2 Inhibitor ABT-199 (GDC-0199)

Background: Acute myeloid leukemia (AML) is characterized by the clonal expansion of immature myeloid cells. AML is treated primarily with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel therapies to achieve higher cure rates with more acceptable toxicities.Bcl-2 family proteins, together with TP53, are the central regulators of apoptosis. Overexpression of Bcl-2 protein is associated with leukemia progression and chemoresistance. We have observed elevated expression of Bcl-2 in AML and recently demonstrated that Bcl-2 inhibition by ABT-199 effectively induced apoptosis in AML (Pan, et.al., Cancer Discovery, 2014). However, resistance to ABT-199 was observed in cells expressing high levels of Mcl-1 or Bcl-xL. Moreover, a recent study showed heterogeneous but overlapping expression of Bcl-2, Mcl-1, and Bcl-xL proteins in 577 AML patient samples (Bogenberger, et. al., Leukemia, 2014).Although common in solid tumors, p53 mutations are relatively rare in AML. However, p53 functions are diminished by overexpression of MDM2 protein, an E3 ubiquitin ligase of p53 and an inhibitor of p53 transactivation. We previously reported MDM2 overexpression in 53% of primary AMLs (Kojima et al., Blood, 2005). Our group also demonstrated that p53 activation by Nutlins, the prototypical MDM2 inhibitors, induced apoptosis and growth inhibition in AML.Rationale: Since p53 activation by MDM2 inhibitors upregulates pro-apoptotic Bcl-2 proteins like NOXA, PUMA, and Bax, which counteract Mcl-1 and Bcl-xL, we hypothesized that the second-generation MDM2 inhibitor RG7388 could overcome AML resistance to Bcl-2-specific ABT-199, and that the combination could synergistically enhance apoptosis in AML.Results: We first demonstrated that RG7388 induced apoptosis exclusively in p53 wild type (wt) cells. RG7388 was essentially ineffective in p53 mutant or null AML cell lines such as HL-60, KG1 and THP1 (48h IC50s > 5 μM). Nonetheless, it showed high potency against p53 wt cell lines (48h IC50s: MOLM13 = 21.7 nM, MV-4-11 = 29.2 nM). Furthermore, stable knockdown of TP53 rendered the wt cell lines completely resistant to RG7388 (IC50s > 5 μM), confirming TP53-specificity. To study if RG7388 was able to overcome inherent resistance to ABT-199, we tested its efficacy on OCI-AML3 cells, which are inherently resistant to ABT-199, AraC and Idarubicin. As a single agent, RG7388 potently killed OCI-AML3 cells (48h IC50 = 148 nM). Importantly, RG7388 was ~20-fold more effective in OCI-AML3 cells than its predecessors Nutlin-3a and RG7112. We also examined the time- and dose-response of RG7388 in several genetically diverse AML cell lines (p53 wt) and found that 100 nM RG7388 was able to induce apoptosis and inhibit cell growth within 12 h.Next we studied whether RG7388 synergizes with ABT-199 to kill the refractory OCI-AML3 cells. A combination index of 0.35 (Chou-Talalay method) indicated a strong synergy between the two compounds. The combination exhibited higher activity in killing OCI-AML3 cells than either agent alone (48h IC50s: ABT-199 = 1680 nM, RG7388 = 148 nM, ABT+RG = 28 nM). Similar synergy was observed in additional AML cell lines and in primary samples. Next, we generated ABT-199 resistant cells by continuous exposure of initially sensitive AML cells to escalating concentrations of ABT-199. While 1000 nM ABT-199 had no effects on the viability of these cells, additional treatment with 30 nM RG7388 effectively killed them. This finding suggested that RG7388 was able to overcome acquired resistance to ABT-199. The mechanisms underlying this resensitization and its synergism with ABT-199 are under investigation using in vitro and in vivo model systems.Conclusions: The novel MDM2 inhibitor RG7388 induces growth arrest and apoptosis selectively in p53 wt AML cells. Importantly, the combination of RG7388 with ABT-199 synergistically induced apoptosis in AML cell lines and primary patient cells, and RG7388 was able to overcome inherent or acquired resistance to ABT-199. Since both Bcl-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical potential. We will report on ongoing experiments with primary AML cells in NSG mice to determine the potential of this combinatorial approach to eliminate AML stem cells. DisclosuresNichols:Roche: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership. Dangl:Roche: Employment, Patents & Royalties. Konopleva:Abbvie: Research Funding. Andreeff:Roche: Research Funding; Abbvie: Research Funding.

Fuse binding protein antagonizes the transcription activity of tumor suppressor protein p53.

BackgroundFUSE binding protein1 (FBP1) is a transactivator of transcription of human c-myc proto-oncogene and expressed mainly in undifferentiated cells. It is also present in differentiated normal cells albeit with very low background. FBP1 is abundantly expressed in the majority of hepatocellular carcinoma tumors and has been implicated in tumor development. Although it down-regulates the expression of proapoptotic p21 protein, it is not known whether FBP1 also interacts and antagonizes the function of tumor suppressor protein p53.MethodsWestern blotting was carried out to detect the expression level of FBP1, p21 and p53, and also p53 regulatory factors, BCCIP and TCTP; real-time quantitative PCR was done to determine the fold change in mRNA levels of target proteins; immunoprecipitation was carried out to determine the interaction of FBP1 with p53, BCCIP and TCTP. Cells stably knockdown for either FBP1; p53 or BCCIP were examined for p53 reporter activity under normal and radiation-induced stress.ResultsFBP1 physically interacted with p53, impairing its transcription activity and reducing p53-mediated sensitivity to cellular stress. Knockdown of FBP1 expression activated p53-mediated response to cellular stress while transient expression of FBP1 in FBP-knockdown cells restored the inhibition of p53 activity. FBP1 not only interacted with both BCCIP and TCTP, which, respectively, function as positive and negative regulators of p53, but also regulated their expression under cellular stress. In FBP knockdown cells, TCTP expression was down-regulated under radiation-induced stress whereas expression of BCCIP and p21 were significantly up-regulated suggesting FBP1 as a potential regulator of these proteins. We hypothesize that the FBP1-mediated suppression of p53 activity may occur via preventing the interaction of p53 with BCCIP as well as by FBP1-mediated regulation of p53 regulatory proteins, TCTP and BCCIP. Since FBP1 suppresses p53 activity and is overexpressed in most HCC tumors, it may have a possible role in tumorigenesis.ConclusionFBP1 physically interacts with p53, functions as a regulator of p53-regulatory proteins (TCTP and BCCIP), and suppresses p53 transactivation activity under radiation-induced cellular stress. Since it is abundantly expressed in most HCC tumors, it may have implication in tumorigenesis and thus may be a possible target for drug development.

Modification of DBC1 by SUMO2/3 is crucial for p53-mediated apoptosis in response to DNA damage.

DBC1 is a major inhibitor of SIRT1, which plays critical roles in the control of diverse cellular processes, including stress response and energy metabolism. Therefore, the DBC1-SIRT1 interaction should finely be regulated. Here we report that DBC1 modification by Small Ubiquitin-like Modifier 2/3 (SUMO 2/3), but not by SUMO1, is crucial for p53 transactivation under genotoxic stress. Whereas etoposide treatment reduced the interaction of DBC1 with SENP1, it promoted that with PIAS3, resulting in an increase in DBC1 sumoylation. Remarkably, the switching from SENP1 to PIAS3 for DBC1 binding was achieved by ATM/ATR-mediated phosphorylation of DBC1. Furthermore, DBC1 sumoylation caused an increase in the DBC1-SIRT1 interaction, leading to the release of p53 from SIRT1 for transcriptional activation. Consistently, SENP1 knockdown promoted etoposide-induced apoptosis, whereas knockdown of PIAS3 or SUMO2/3 and overexpression of sumoylation-deficient DBC1 mutant inhibited it. These results establish the role of DBC1 sumoylation in the promotion of p53-mediated apoptosis in response to genotoxic stress.

Abstract 4071: SAH-p53-mediated inhibition of cell migration via alteration of actin dynamics

Abstract The mutation of p53 is critical for the increased metastatic potential of cancer cells. As such, it is essential not only to understand the mechanisms underlying the functional implications of p53 mutation, but also to identify ways to regulate their impact on cancer cell physiology. I have found that a hydrocarbon-stapled p53 transactivation domain peptide (SAH-p53) designed to disrupt the interaction of p53 with its negative regulators HDM2 and HDMX retains its function in cells harboring a mutant TP53. A common gain-of-function activity of the mutant p53 protein (mtp53) is the enhancement of cell migration and invasion; however, I found that SAH-p53 has the ability to inhibit the migration and invasion of HS578T and MDA-MB-231 breast cancer cells (mtp53), while exhibiting no change in proliferative ability at the same dose. This effect was accompanied by sustained alterations in actin stress fiber dynamics and inhibition of focal adhesion assembly and signaling. Furthermore, treatment of MDA-MB-231 cells with various growth factors (EGF, TGF-β, PDGF, HGF) was unable to rescue the migratory ability of cells in the presence of SAH-p53. The combination of SAH-p53 and TGF-β resulted in a decrease in the total expression levels of both EGFR and FAK. Moreover, it was shown that the combined treatment of SAH-p53 and TGF-β enhanced the localization of EGFR to early endosomes and eventually to lysosomes, prompting the use of fluorophore-capped SAH-p53 to track the localization of the peptide in relation to EGFR. It was determined that over time SAH-53 localizes to vesicles containing EGFR, but not other RTKs (ie. PDGFRβ). SAH-p53 was able to retain its ability to inhibit migration in the absence of functional HDMX/HDM2 or mutant p53, suggesting the possibility of an alternative target. We are currently using a photoactivatable and capped SAH-p53 that can cross-link to a target protein in the presence of UV in order to determine the identity of the target of SAH-p53 responsible for the anti-migratory effects seen in mutant p53- or p53-null cells. These data show that the use of a stapled peptide provides a novel avenue to study the molecular interactions that drive metastatic behavior in cancer cells. Citation Format: Bethanie L. Morrison, Amanda L. Whiting, Federico Bernal. SAH-p53-mediated inhibition of cell migration via alteration of actin dynamics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2014-4071

Abstract 3393: Nuclear receptor Nurr1 mediates cell proliferation and is involved in a p53-microRNA-34 regulatory network

Abstract Nuclear receptor (NR) subfamily 4 member 2 (NR4A2), also known as Nurr1, has been shown to have oncogenic properties, in which it can mediate cell transformation, migration and drug resistance. However, the manner in which Nurr1 mediates these cancer-promoting features is not completely understood, but research implicates p53 as a potential intermediary. In this study, we show that Nurr1 enhances the proliferative rate of colorectal cancer cells. This increase in cell growth is accompanied by an upregulation of cyclin D3, a putative target gene of Nurr1. To understand how Nurr1 itself is regulated, we performed a reporter screen to identify microRNA regulators of Nurr1's 3’UTR. We identified miR-34c as a putative regulator of Nurr1 and this direct regulation was confirmed through site-directed mutagenesis of the predicted seed region. We next determined if p53 activation could affect Nurr1 expression through transcriptional activation of miR-34. Using Nutlin-3a to activate p53, we found that Nurr1 expression decreased in a dose response manner. Lastly, we sought to determine if Nurr1 could attenuate p53 activation. We found that overexpression of Nurr1 in colorectal cancer cells caused an attenuation of p53 transactivation of target genes following Nutlin-3a treatment. Our results suggest that a p53-miR-34-Nurr1 regulatory network exists, which would allow p53 to maintain Nurr1 in a suppressed state through miR-34-mediated regulation and prevent tumorigenesis. In cases of p53 mutation or Nurr1 overexpression, this network would be compromised, allowing Nurr1 to enhance tumorigenesis or drug resistance. Citation Format: Jordan A. Beard, Justin L. Hills, Alexa Tenga, Apana A. Takwi, Taosheng Chen. Nuclear receptor Nurr1 mediates cell proliferation and is involved in a p53-microRNA-34 regulatory network. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3393. doi:10.1158/1538-7445.AM2014-3393

Abstract 3234: Covalent capture of protein binding partners using an azide-tagged, photo-reactive stapled alpha helical p53 peptide

Abstract Mutations in the DNA binding region of tumor suppressor p53 render it transcriptionally inactive (dominant negative) but can also manifest in transcriptionally-independent gain-of-function (GOF) effects, possibly due to mutp53 interacting with other, as yet, unknown targets. Identification of these targets would help to elucidate the pathways and vulnerabilities involved in mutp53 GOF. Covalently linking a known handle to these unknown proteins is one method to aid in their isolation and identification. A hydrocarbon-stapled alpha helical peptide of the p53 transactivation domain (residues 14-29), SAH-p53-8, has been shown to strongly interact with known p53 targets that utilize this domain for binding (HDM2, Kd = 55 nM, HDMX, Kd = 2.3 nM). Substitution of a key interacting residue for the unnatural amino acid benzoylphenylalanine (Bpa) results in a stapled peptide that retains the biochemical properties of SAH-p53-8 (affinities of 38.1 nM and 58.3 nM, respectively) and can covalently capture known targets via photochemical reaction. The introduction of an azido group - a versatile, biochemically-inert capping group - to the N-terminus of this photo-reactive peptide allows for the attachment of various tags to the covalently bound protein(s) via a copper-catazlyzed azide alkyne cycloaddition. Tags include both fluorophores for visualization and affinity tags for protein isolation. This poster will cover the biochemical proof-of-principle of this protein isolation method and exploration into more complex cell lysate environments. Citation Format: Amanda L. Whiting, Joe J. Mitala, Kathryn M. Headley, Joe Reilly, Bethanie L. Morrison, Kevin A. Murray, Federico Bernal. Covalent capture of protein binding partners using an azide-tagged, photo-reactive stapled alpha helical p53 peptide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2014-3234

Abstract 2459: The oncogenic activity of MDMx is associated with physical interaction and suppression of retinoblastoma protein

Abstract The aim of this study is to investigate the role of MDMx (or MDM4) on the regulation of retinoblastoma protein (Rb). Inactivation of Rb plays a critical role in the development of human malignancies. We have previously shown that MDM2, an ubiquitin E3 ligase and a major negative regulator of p53, binds to and promotes proteasome-mediated degradation of Rb independent of p53. MDMX, a homolog of MDM2, also binds to and inhibits p53 transactivation activity, yet it does not possess an ubiquitin E3 ligase activity. In this study, we show that MDMX binds to and promotes proteasome-mediated degradation of Rb in a MDM2-dependent manner. The C-terminal Ring domain of MDMX binds to the Rb C-pocket and enhances MDM2-Rb interaction. Knockdown of MDMX induces Rb protein accumulation, cell cycle G1 arrest, and premature cellular senescence, which can be reverted by simultaneous knockdown of Rb. Furthermore, ablation of MDMX significantly suppresses tumor growth, concomitant with Rb accumulation, in an animal xenograft model. Together, these results demonstrate that MDMX possesses oncogenic activity associated with suppression of Rb and suggest that both MDM2 and MDMX can be targets for cancer therapy. Note: This abstract was not presented at the meeting. Citation Format: Zhi-Xiong Xiao, Haibo Zhang, Yujun Zhang. The oncogenic activity of MDMx is associated with physical interaction and suppression of retinoblastoma protein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2459. doi:10.1158/1538-7445.AM2014-2459

Protein grafting of p53TAD onto a leucine zipper scaffold generates a potent HDM dual inhibitor.

Reactivation of the p53 pathway by a potential therapeutic antagonist, which inhibits HDM2 and HDMX, is an attractive strategy for drug development in oncology. Developing blockers towards conserved hydrophobic pockets of both HDMs has mainly focused on small synthetic compounds; however, this approach has proved challenging. Here we describe an approach to generate a potent HDM dual inhibitor, p53LZ2, by rational protein grafting of the p53 transactivation domain onto a homodimeric leucine zipper. p53LZ2 shows tight binding affinity to both HDMs compared with wild-type p53 in vitro. X-ray crystallographic, comparative modelling and small-angle X-ray scattering studies of p53LZ2-HDM complexes show butterfly-shaped structures. A cell-permeable TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro. Thus, p53LZ2, designed by rational grafting, shows a potential therapeutic approach against cancer.