Cooperative interactions between p53 and NFkappaB enhance cell plasticity.

The p53 and NFkappaB sequence-specific transcription factors play crucial roles in cell proliferation and survival with critical, even if typically opposite, effects on cancer progression. To investigate a possible crosstalk between p53 and NFkappaB driven by chemotherapy-induced responses in the context of an inflammatory microenvironment, we performed a proof of concept study using MCF7 cells. Transcriptome analyses upon single or combined treatments with doxorubicin (Doxo, 1.5muM) and the NFkappaB inducer TNF-alpha (TNFalpha, 5ng/ml) revealed 432 up-regulated (log2 FC> 2), and 390 repressed genes (log2 FC< -2) for the Doxo+TNFalpha treatment. 239 up-regulated and 161 repressed genes were synergistically regulated by the double treatment. Annotation and pathway analyses of Doxo+TNFalpha selectively up-regulated genes indicated strong enrichment for cell migration terms. A panel of genes was examined by qPCR coupled to p53 activation by Doxo, 5-Fluoruracil and Nutlin-3a, or to p53 or NFkappaB inhibition. Transcriptome data were confirmed for 12 of 15 selected genes and seven (PLK3, LAMP3, ETV7, UNC5B, NTN1, DUSP5, SNAI1) were synergistically up-regulated after Doxo+TNFalpha and dependent both on p53 and NFkappaB. Migration assays consistently showed an increase in motility for MCF7 cells upon Doxo+TNFalpha. A signature of 29 Doxo+TNFalpha highly synergistic genes exhibited prognostic value for luminal breast cancer patients, with adverse outcome correlating with higher relative expression. We propose that the crosstalk between p53 and NFkappaB can lead to the activation of specific gene expression programs that may impact on cancer phenotypes and potentially modify the efficacy of cancer therapy

TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection–associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation

Safety and Feasibility of MitraClip Implantation in Patients with Acute Mitral Regurgitation after Recent Myocardial Infarction and Severe Left Ventricle Dysfunction

Patients with severe mitral regurgitation (MR) after myocardial infarction (MI) have an increased risk of mortality. Transcatheter mitral valve repair may therefore be a suitable therapy. However, data on clinical outcomes of patients in an acute setting are scarce, especially those with reduced left ventricle (LV) dysfunction. We conducted a multinational, collaborative data analysis from 21 centers for patients who were, within 90 days of acute MI, treated with MitraClip due to severe MR. The cohort was divided according to median left ventricle ejection fraction (LVEF)-35%. Included in the study were 105 patients. The mean age was 71 ± 10 years. Patients in the LVEF \u3c 35% group were younger but with comparable Euroscore II, multivessel coronary artery disease, prior MI and coronary artery bypass graft surgery. Procedure time was comparable and acute success rate was high in both groups (94% vs. 90%, p = 0.728). MR grade was significantly reduced in both groups along with an immediate reduction in left atrial V-wave, pulmonary artery pressure and improvement in New York Heart Association (NYHA) class. In-hospital and 1-year mortality rates were not significantly different between the two groups (11% vs. 7%, p = 0.51 and 19% vs. 12%, p = 0.49) and neither was the 3-month re-hospitalization rate. In conclusion, MitraClip intervention in patients with acute severe functional mitral regurgitation (FMR) due to a recent MI in an acute setting is safe and feasible. Even patients with severe LV dysfunction may benefit from transcatheter mitral valve intervention and should not be excluded

p53 Transactivation and the Impact of Mutations, Cofactors and Small Molecules Using a Simplified Yeast-Based Screening System

The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. promoter, ii) single copy, chromosomally located p53-responsive and control luminescence reporters, iii) enhanced chemical uptake using modified ABC-transporters, iv) small-volume formats for treatment and dual-luciferase assays, and v) opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1.We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins

p53 Functional Interactions: the Study of a New Crosstalk with Estradiol Pathway in Transcriptional Responses to Chemotherapeutics

BACKGROUND: Objective of this thesis has been the analysis of the sequence specific transcription factor p53, a critical tumor suppressor protein, specifically, the crosstalk (or functional interactions) with other transcription factors, namely, the estrogen receptors, and the modeling in reconstituted assays of the interaction of p53 with positive and negative cofactors (e.g. MDM4 and 53BP1) and the impact of small molecules, including chemotherapeutic drugs, on such interactions. Previous reports have revealed a complex, often negative, crosstalk between p53 and estrogen receptors (ERs) related in part to the physical interaction between the two proteins. An example of transcriptional cooperation mediated by cognate, non-canonical cis-elements was instead discovered for the angiogenesis related VEGFR1, FLT1 promoter. MAIN TASK: Transcriptional cooperation between p53 and ERs was sought out on a global scale using the human breast adenocarcinoma MCF7 cells as a model and transcriptome analyses. Cells were subjected to single or combinatorial treatments with the chemotherapeutic agent doxorubicin (able to induce p53 protein stabilization) and the ER ligand 17β-estradiol (E2). 201 differentially expressed genes, that showed limited responsiveness to either doxorubicin treatment or ER ligand alone, but were up-regulated in a greater than additive manner following combined treatment were identified. Among sixteen genes chosen for validation using quantitative real-time PCR (qPCR), seven (INPP5D, TLR5, KRT15, EPHA2, GDNF, NOTCH1, SOX9) were confirmed to be novel direct targets of p53, based on responses in stable MCF7 clone cells silenced for p53, or cooperative targets of p53 and ER. Based on exposure to 5-fuorouracil (another genotoxic drug) and nutlin-3a (a non-genotoxic p53-specific activator), the combined response identified genes that were consistently regulated, although with different kinetics (e.g. INPP5D, CDH26, KRT15), while others (e.g. TLR5, SOX9) were treatment selective. Promoter pattern searches and chromatin IP experiments for the INPP5D, TLR5, KRT15 genes were also performed to interrogate a direct, cis-mediated p53 and ERs regulation. While these analyses confirmed the identification of novel direct p53 targets, the important contribution of ER in their transcriptional modulation and the role of non-canonical response elements, the correlation between occupancy levels and gene expression varied. SECONDARY TASK: Using a newly developed miniaturized yeast-based assay, functional interactions between p53 and its regulators MDM4 and 53BP1 was investigated. MDM4 was confirmed as a p53 negative regulator and the impact of nutlin-3a or RITA (apoptosis inducer through p53 binding) on the p53-MDM4 interaction was explored. Instead, no stimulatory effect of the p53 co-activator 53BP1 was detected. CONCLUSIONS: Collectively, the results indicate that combinatorial activation of p53 and ER can induce novel gene expression programs which have implications for cell-cell communications, adhesion, cell differentiation, development and inflammatory responses as well as cancer treatments. The yeast-based assay represents a versatile tool to study p53 interactions with cofactors

Abstract LB-170: Genotoxic stress-dependent, p53-directed post-transcriptional controls of the VEGFR-1, FLT1 gene

Abstract We recently established that a C&amp;gt;T SNP in the promoter of the VEGFR-1 gene, commonly known as FLT1, generates a half-site p53 response element (RE-T) that results in p53 responsiveness of the promoter. It was also shown that p53 is required but not sufficient for FLT1 transactivation and that there is cooperative interaction with ligand-bound Estrogen Receptors (ERs) via two ER half-site (EREs) located near the RE-T. ER levels, specific ligands and genotoxic stresses were shown to influence the coordinated regulation of the FLT1-T promoter. We have now asked whether activation of p53 by genotoxic stress could also regulate FLT1 at post-transcriptional levels. The FLT1 transcript can undergo alternative splicing resulting either in a membrane-bound- (mFLT1) or in a truncated soluble-form (sFLT1) that appears to act as decoy receptor, where a portion of intron 13 is retained in the mRNA introducing a premature STOP codon and an alternative 3′UTR. We measured by qPCR the expression levels of mFLT1 and sFLT1 transcripts in cell lines differing for the promoter SNP and p53 status: HCT116 (C/T, p53 wt+/+or p53 null−/−), GIMEN (C/T, p53 wt), MCF7 (C/C, p53 wt). Surprisingly, we observed that the relative abundance of mFLT1 and sFLT1 was differentially affected by doxorubicin treatment (doxo) in that mFLT1 was up-regulated in C/T heterozygous p53 wt cells, as expected, while sFLT1 levels did not change or were even reduced, depending on the cell line. Notably, ectopic expression of p53 in HCT116−/− led to the same specific increase in mFLT1/sFLT1 expression ratio. On the contrary, treatment with 5FU, while effective at activating p53, led to a selective strong upregulation of sFLT1. This effect was seen also in the MCF7 C/C cell line, but appeared to be dependent on the presence of p53 wt, based on results in HCT116−/−. As the mFLT1 and sFLT1 transcripts contain different 3′UTRs, we investigated using luciferase-based reporter vectors whether these regulatory sequences could underlie the differential response to p53 activation. Results indicated that doxo treatment in HCT116+/+ selectively increased mFLT1–3′UTR stability. 5FU had no differential impact in this assay. We also developed an FLT1 minigene containing exon 13, exon 14 and part of the intron 13 as intervening sequence in a dual luciferase vector. Interestingly, in HCT116+/+ cells transfected with the minigene treatment with 5FU but not doxo led to a shift in the splicing pattern toward sFLT1. Treatment with Nutlin, a molecule disrupting p53-MDM2 interaction, has a similar impact as 5FU. The effect of genotoxic stress-dependent, p53-directed changes in FLT1 transcripts' balance on FLT1-induced signalling is currently being evaluated, using the specific ligand PlGF. Collectively our emerging results are revealing that, in response to specific stimuli p53 can regulate not only FLT1 transcription, but also its mRNA maturation and stability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-170. doi:10.1158/1538-7445.AM2011-LB-170</jats:p

Alterations in chromatin at antigen receptor loci define lineage progression during B lymphopoiesis

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes seem to be regulated independently and can occur prior to lineage commitment. Large-scale changes in chromatin accessibility occur without significant change in nucleosome density and represent key features of AgR loci not previously described. We further identify local dynamic repositioning of individual RSS-associated nucleosomes at IgH and Igκ loci while they become primed for recombination during B cell commitment. These changes in chromatin at AgR loci are regulated in a locus-, lineage-, and stage-specific manner during B lymphopoiesis, serving either to facilitate or to impose a barrier to V(D)J recombination. We suggest that local and global changes in chromatin openness in concert with nucleosome occupancy and placement of histone modifications facilitate the temporal order of AgR recombination. Our data have implications for the organizing principles that govern assembly of these large loci as well as for mechanisms that might contribute to aberrant V(D)J recombination and the development of lymphoid tumors.</jats:p