Anthrax Edema Toxin Modulates PKA- and CREB-Dependent Signaling in Two Phases

Background: Anthrax edema toxin (EdTx) is an adenylate cyclase which operates in the perinuclear region of host cells. However, the action of EdTx is poorly understood, especially at molecular level. The ability of EdTx to modulate cAMPdependent signaling was studied in Jurkat T cells and was compared with that of other cAMP-rising agents: Bordetella pertussis adenylate cyclase toxin, cholera toxin and forskolin. Methodology/Principal Findings: EdTx caused a prolonged increase of the intracellular cAMP concentration. This led to nuclear translocation of the cAMP-dependent protein kinase (PKA) catalytic subunit, phosphorylation of cAMP response element binding protein (CREB) and expression of a reporter gene under control of the cAMP response element. Neither p90 ribosomal S6 kinase nor mitogen- and stress-activated kinase, which mediate CREB phosphorylation during T cell activation, were involved. The duration of phospho-CREB binding to chromatin correlated with the spatio-temporal rise of cAMP levels. Strikingly, EdTx pre-treated T cells were unresponsive to other stimuli involving CREB phosphorylation such as addition of forskolin or T cell receptor cross-linking. Conclusions/Significance: We concluded that, in a first intoxication phase, EdTx induces PKA-dependent signaling, which culminates in CREB phosphorylation and activation of gene transcription. Subsequently CREB phosphorylation is impaired and therefore T cells are not able to respond to cues involving CREB. The present data functionally link the perinuclea

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

The β2-adrenergic receptor is a molecular switch for neuroendocrine transdifferentiation of prostate cancer cells

The incidence of treatment-related neuroendocrine (NE) prostate cancer (t-NEPC) is rising as more potent drugs targeting the androgen signaling axis are clinically implemented. Neuroendocrine transdifferentiation (NEtD), an putative initial step in t-NEPC development, is induced by androgen deprivation therapy (ADT) or anti-androgens, and by activation of the β2-adrenergic receptor (ADRB2) in prostate cancer cell lines. Thus, understanding whether ADRB2 is involved in ADT-initiated NEtD may assist in developing treatment strategies that can prevent or reverse t-NEPC emergence, thereby prolonging therapeutic responses. Here we found that in primary, treatment-naïve prostate cancers, ADRB2 mRNA was positively correlated with expression of luminal differentiation markers, and ADRB2 protein levels were inversely correlated with Gleason grade. ADRB2 mRNA was upregulated in metastatic prostate cancer, and progressively downregulated during androgen deprivation therapy (ADT) and t-NEPC emergence. In androgen-deprivated medium, high ADRB2 was required for LNCaP cells to undergo NEtD, measured as increased neurite outgrowth and expression of neuron differentiation and NE genes. ADRB2 overexpression induced an NE-like morphology in both AR positive and -negative prostate cancer cell lines. ADRB2 downregulation in LNCaP cells increased canonical Wnt signaling, and GSK3α/β inhibition reduced the expression of neuron differentiation and NE genes. In LNCaP xenografts, more pronounced castration-induced NEtD was observed in tumors derived from high than low-ADRB2 cells. In conclusion, high ADRB2 expression is required for ADT-induced NEtD, characterized by ADRB2 downregulation and t-NEPC emergence. Implications: This data suggest a potential application of β-blockers to prevent cancer cells committed to a neuroendocrine lineage from evolving into t-NEPC

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E(1). However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities

Direct AKAP-mediated protein-protein interactions as potential drug targets

A-kinase-anchoring proteins (AKAPs) are a diverse family of about 50 scaffolding proteins. They are defined by the presence of a structurally conserved protein kinase A (PKA)-binding domain. AKAPs tether PKA and other signalling proteins such as further protein kinases, protein phosphatases and phosphodiesterases by direct protein-protein interactions to cellular compartments. Thus, AKAPs form the basis of signalling modules that integrate cellular signalling processes and limit these to defined sites. Disruption of AKAP functions by gene targeting, knockdown approaches and, in particular, pharmacological disruption of defined AKAP-dependent protein-protein interactions has revealed key roles of AKAPs in numerous processes, including the regulation of cardiac myocyte contractility and vasopressin-mediated water reabsorption in the kidney. Dysregulation of such processes causes diseases, including cardiovascular and renal disorders. In this review, we discuss AKAP functions elucidated by gene targeting and knockdown approaches, but mainly focus on studies utilizing peptides for disruption of direct AKAP-mediated protein-protein interactions. The latter studies point to direct AKAP-mediated protein-protein interactions as targets for novel drugs