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

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

Reactivation of TAp73 tumor suppressor by protoporphyrin IX, a metabolite of aminolevulinic acid, induces apoptosis in TP53-deficient cancer cells

Abstract Background The p73 protein is a tumor suppressor that shares structural and functional similarity with p53. p73 is expressed in two major isoforms; the TA isoform that interacts with p53 pathway, thus acting as tumor suppressor and the N-terminal truncated ΔN isoform that inhibits TAp73 and p53 and thus, acts as an oncogene. Results By employing a drug repurposing approach, we found that protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid applied in photodynamic therapy of cancer, stabilizes TAp73 and activates TAp73-dependent apoptosis in cancer cells lacking p53. The mechanism of TAp73 activation is via disruption of TAp73/MDM2 and TAp73/MDMX interactions and inhibition of TAp73 degradation by ubiquitin ligase Itch. Finally, PpIX showed potent antitumor effect and inhibited the growth of xenograft human tumors in mice. Conclusion Our findings may in future contribute to the successful repurposing of PpIX into clinical practice