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

Tolerability of new antiepileptic drugs:a network meta-analysis

Objective: The objective of this study was to perform a comparative assessment of tolerability of all licensed new antiepileptic drugs (AEDs) through a network meta-analysis (NMA) including all placebo-controlled double-blind clinical trials (RCTs) in all conditions in which these drugs have been tested. Methods: NMA with a frequentist approach was used to compare proportions of patients withdrawing because of adverse events (AEs). Analyses were conducted for all therapeutic doses pooled and specifically for high therapeutic doses. Patients treated with non-therapeutic doses of each drug were excluded. Results: A total of 195 RCTs were included in the current analysis, comprising a total of 28,013 patients treated with AEDs and 17,908 patients treated with placebo. RCTs included in the analysis were 8 for brivaracetam; 5 for eslicarbazepine; 22 for gabapentin; 7 for lacosamide; 14 for levetiracetam; 14 for lamotrigine; 6 for oxcarbazepine; 9 for perampanel; 50 for pregabalin; 5 for tiagabine; 36 for topiramate; 7 for zonisamide; 4 for gabapentin-extended formulation (ER); 2 each for levetiracetam-ER, lamotrigine-ER, and topiramate-ER; and 1 each for oxcarbazepine-ER and pregabalin-ER. Brivaracetam, gabapentin, gabapentin-ER, and levetiracetam had a significantly lower withdrawal rate compared to several other AEDs, while eslicarbazepine, lacosamide, oxcarbazepine, and topiramate had a higher withdrawal rate. Perampanel, lamotrigine, pregabalin, tiagabine, and zonisamide showed an intermediate pattern of tolerability. Additional analysis has been conducted through selection of highly recommended doses for each drug. This analysis has roughly confirmed results of head to head comparisons of the all-dose analysis, with some exceptions. A further analysis has been conducted after exclusion of RCTs in which patients were allocated to the therapeutic dose of the experimental drug without titration, and it failed to show clinically important differences. Significance: Relevant differences in short-term tolerability of AEDs have been observed between AEDs. Brivaracetam, gabapentin, and levetiracetam show the best tolerability profile while other AEDs are at higher risk for intolerable adverse effects

Antiepileptic drugs’ tolerability and safety – a systematic review and meta-analysis of adverse effects in dogs

<p>Various anti-epileptic drugs (AEDs) are used for the management of idiopathic epilepsy (IE) in dogs. Their safety profile is an important consideration for regulatory bodies, owners and prescribing clinicians. However, information on their adverse effects still remains limited with most of it derived from non-blinded non-randomized uncontrolled trials and case reports.</p><p><span>This poster won third place, which was presented at the Veterinary Evidence Today conference, Edinburgh November 1-3, 2016. </span></p><br /> <img src="https://www.veterinaryevidence.org/rcvskmod/icons/oa-icon.jpg" alt="Open Access" /

Translational control mechanisms in the p53 response network

The sequence-specific transcription factor p53 is considered a master gene of cellular responses to homeostasis changes. It is also a prominent tumor suppressor gene with the title of “guardian of the genome”. The increasing number of transcriptome analyses in cell lines treated with different agents activating p53, continues to add complexity to the vast transcriptional networks p53 regulates. To investigate mRNA translational control as an additional dimension of p53-directed gene expression responses, we performed translatome analyses upon its activation either by different agents or cellular contexts. Considered as a proxy for the proteome, the translatome allows us to characterize the translational status of each mRNA, independently from transcriptional modulations, and to evaluate the implications or correlations of changes in relative mRNA translation efficiencies with the phenotypic outcome. We first performed treatment-specific translatome profiling in MCF7 cells upon Doxorubicin and Nutlin-3a treatments. Among translated genes, we detected the presence of translationally enhanced mRNAs with a virtually absent transcriptional modulation; those genes were enriched for apoptotic functions, suggesting that the apoptotic phenotype might be controlled not only at the transcriptional, but also at the translational level. Seeking mechanisms underlying the mRNAs translational rate upon p53 activation, we identified the modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets, modulated at the translational level in a p53-dependent manner. In detail, we demonstrated the contribution of at least two p53-dependent translational mechanisms related to YBX1 translational repression, suggesting the presence of a controlled regulon at the crossroad of YBX1 mRNA translation. Given our finding that apoptotic genes appear to be controlled by p53 also at the translational level, we decided to explore whether mRNAs translational control mechanisms are indeed an additional checkpoint to the phenotype. To this aim, we performed a cell-type specific translatome study upon Nutlin-3a treatment, a drug with evident therapeutic prospective. SJSA1, HCT116 and MCF7 cells were chosen as they exhibit different cellular responses to Nutlin-3A (cell cycle arrest, apoptosis, or both, respectively). Our preliminary data suggests that translational modulation can affect the complex process of cell fate choice upon p53 activation. Indeed, a lack of overlap among genes differentially modulated at the translational level was evident. Motif search analysis at the 5’- and 3’-UTR of those genes highlighted the presence of different motifs in the three cell lines and the specific correlation of a C-rich motif with the apoptotic phenotype. Preliminary data on this motif will be presented and discussed. Two independent projects will be presented as appendixes, both of them related to the general idea that more than one factor may determine the p53 response. Starting from the analysis of possible p53 interactions with other transcriptional co-factors, we investigated the cooperative interaction between p53 and NFκB. For the second project, combining data previously obtained by means of yeast-based p53 transactivation assays, we developed an algorithm, p53retriever, to scan DNA sequences and thus identify p53 response elements and classify them based on their transactivation potential

Translation control can shape TP53-dependent cell fate

The search for mechanisms underlying different cellular responses to the treatment with Nutlin-3, an MDM2 inhibitor that unleashes p53, revealed a translational control mechanism involving the RNA binding proteins PCBP2 and, particularly, DHX30. Sifting through a multi-functional p53-dependent transcriptional output, this translational control can modulate the activation of cell death pathways

Live imaging of mRNA using RNA-stabilized fluorogenic proteins

Fluorogenic RNA aptamers bind and activate the fluorescence of otherwise nonfluorescent dyes. However, fluorogenic aptamers are limited by the small number of fluorogenic dyes suitable for use in live cells. In this communication, fluorogenic proteins whose fluorescence is activated by RNA aptamers are described. Fluorogenic proteins are highly unstable until they bind RNA aptamers inserted into messenger RNAs, resulting in fluorescent RNA–protein complexes that enable live imaging of mRNA in living cells

Identification of the m6Am Methyltransferase PCIF1 Reveals the Location and Functions of m6Am in the Transcriptome

International audiencemRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2'-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am's function