Wolfgang Link

Understanding FOXO, new views on old transcription factors.

FOXO proteins are evolutionarily conserved transcription factors implicated in several fundamental cellular processes, functioning as end-point for transcriptional programs involved in apoptosis, stress response and longevity. Abrogation of FOXO function is very frequent in human cancer, therefore the mechanisms of regulation of the FOXO proteins are receiving increasing attention in cancer research. The FOXO proteins integrate regulatory inputs from a variety of upstream signaling pathways, most importantly in response to growth factor and stress signalling. Recently, FOXO factors have been established as tumor suppressors, promoting the transcription of pro-apoptotic molecules like FasL and Bim when the PI3K/Akt pathway is downregulated due to nutrient or serum starvation and cellular stress. Therefore, understanding the modulation of FOXO transcription factors will allow the design of new compounds with antitumor potential.

DREAM is a Ca2+-regulated transcriptional repressor

Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function,. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved inmemory acquisition and pain,, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAMs ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.

The PTEN/PI3K/AKT Signalling Pathway in Cancer, Therapeutic Implications

PTEN/PI3K/AKT constitutes an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. PTEN is a dual protein/lipid phosphatase which main substrate is the phosphatidyl-inositol,3,4,5 triphosphate (PIP3), the product of PI3K. Increase in PIP3 recruits AKT to the membrane where it is activated by other kinases also dependent on PIP3. Many components of this pathway have been described as causal forces in cancer. PTEN activity is lost by mutations, deletions or promoter methylation silencing at high frequency in many primary and metastatic human cancers. Germ line mutations of PTEN are found in several familial cancer predisposition syndromes. Activating mutations which have been reported for PI3K and AKT, in tumours are able to confer tumourigenic properties in several cellular systems. Additionally, the binding of PI3K to oncogenic ras is essential for the transforming properties of ras. In summary, the data strongly support the view of the PTEN/PI3K/AKT pathway as an important target for drug discovery.

High content screening: seeing is believing

High content screening (HCS) combines the efficiency of high-throughput techniques with the ability of cellular imaging to collect quantitative data from complex biological systems. HCS technology is integrated into all aspects of contemporary drug discovery, including primary compound screening, post-primary screening capable of supporting structure-activity relationships, and early evaluation of ADME (absorption, distribution, metabolism and excretion)/toxicity properties and complex multivariate drug profiling. Recently, high content approaches have been used extensively to interrogate stem cell biology. Despite these dramatic advances, a number of significant challenges remain related to the use of more biology- and disease-relevant cell systems, the development of informative reagents to measure and manipulate cellular events, and the integration of data management and informatics.

Protein localization in disease and therapy

The eukaryotic cell is organized into membrane-covered compartments that are characterized by specific sets of proteins and biochemically distinct cellular processes. The appropriate subcellular localization of proteins is crucial because it provides the physiological context for their function. In this Commentary, we give a brief overview of the different mechanisms that are involved in protein trafficking and describe how aberrant localization of proteins contributes to the pathogenesis of many human diseases, such as metabolic, cardiovascular and neurodegenerative diseases, as well as cancer. Accordingly, modifying the disease-related subcellular mislocalization of proteins might be an attractive means of therapeutic intervention. In particular, cellular processes that link protein folding and cell signaling, as well as nuclear import and export, to the subcellular localization of proteins have been proposed as targets for therapeutic intervention. We discuss the concepts involved in the therapeutic restoration of disrupted physiological protein localization and therapeutic mislocalization as a strategy to inactivate disease-causing proteins.

Long live FOXO: unraveling the role of FOXO proteins in aging and longevity

Aging constitutes the key risk factor for age‐related diseases such as cancer and cardiovascular and neurodegenerative disorders. Human longevity and healthy aging are complex phenotypes influenced by both environmental and genetic factors. The fact that genetic contribution to lifespan strongly increases with greater age provides basis for research on which “protective genes” are carried by long‐lived individuals. Studies have consistently revealed FOXO (Forkhead box O) transcription factors as important determinants in aging and longevity. FOXO proteins represent a subfamily of transcription factors conserved from Caenorhabditis elegans to mammals that act as key regulators of longevity downstream of insulin and insulin‐like growth factor signaling. Invertebrate genomes have one FOXO gene, while mammals have four FOXO genes: FOXO1, FOXO3, FOXO4, and FOXO6. In mammals, this subfamily is involved in a wide range of crucial cellular processes regulating stress resistance, metabolism, cell cycle arrest, and apoptosis. Their role in longevity determination is complex and remains to be fully elucidated. Throughout this review, the mechanisms by which FOXO factors contribute to longevity will be discussed in diverse animal models, from Hydra to mammals. Moreover, compelling evidence of FOXOs as contributors for extreme longevity and health span in humans will be addressed.

DREAM-αCREM Interaction via Leucine-Charged Domains Derepresses Downstream Regulatory Element-Dependent Transcription

ABSTRACT Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, αCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of αCREM is not required for the interaction, but phospho-αCREM shows higher affinity for DREAM. The interaction with αCREM is independent of the Ca2+-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both αCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca2+.

Interleukin 3-dependent activation of DREAM is involved in transcriptional silencing of the apoptotic hrk gene in hematopoietic progenitor cells

The apoptotic protein Hrk is expressed in hematopoietic progenitors after growth factor deprivation. Here we identify a silencer sequence in the 3′ untranslated region of the hrk gene that binds to the transcriptional repressor DREAM in interleukin‐3 (IL‐3)‐dependent hematopoietic progenitor cells, and abrogates the expression of reporter genes when located downstream of the open reading frame. In addition, the binding of DREAM to the hrk gene is reduced or eliminated when cells are cultured in the absence of IL‐3 or treated with a calcium ionophore or a phosphatidylinositol 3‐kinase‐specific inhibitor, suggesting that both calcium mobilization and phosphorylation can regulate the transcriptional activity of DREAM. Furthermore, we have shown that DREAM is phosphorylated by a phosphatidylinositol 3‐kinase‐dependent, but Akt‐independent pathway. In all cases, loss of the DREAM–DNA binding complex was correlated with increased levels of Hrk and apoptosis. These data suggest that IL‐3 may trigger the activation of DREAM through different signaling pathways, which in turn binds to a silencer sequence in the hrk gene and blocks transcription, avoiding inappropriate cell death in hematopoietic progenitors.

Day-Night Changes in Downstream Regulatory Element Antagonist Modulator/Potassium Channel Interacting Protein Activity Contribute to Circadian Gene Expression in Pineal Gland

The molecular mechanisms controlling the oscillatory synthesis of melatonin in rat pineal gland involve the rhythmic expression of several genes including arylalkylamine N-acetyltransferase (AA-NAT), inducible cAMP early repressor (ICER), and Fos-related antigen-2 (fra-2). Here we show that the calcium sensors downstream regulatory element antagonist modulator/potassium channel interacting protein (DREAM/KChIP)-3 and KChIP-1, -2 and -4 bind to downstream regulatory element (DRE) sites located in the regulatory regions of these genes and repress basal and induced transcription from ICER, fra-2 or AA-NAT promoters. Importantly, we demonstrate that the endogenous binding activity to DRE sites shows day-night oscillations in rat pineal gland and retina but not in the cerebellum. The peak of DRE binding activity occurs during the day period of the circadian cycle, coinciding with the lowest levels of fra-2, ICER, and AA-NAT transcripts. We show that a rapid clearance of DRE binding activity during the entry in the night period is related to changes at the posttranscriptional level of DREAM/KChIP. The circadian pattern of DREAM/KChIP activity is maintained under constant darkness, indicating that an endogenous clock controls DREAM/KChIP function. Our data suggest involvement of the family of DREAM repressors in the regulation of rhythmically expressed genes engaged in circadian rhythms.

Chemical Interrogation of FOXO3a Nuclear Translocation Identifies Potent and Selective Inhibitors of Phosphoinositide 3-Kinases

Activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is one the most frequent genetic events in human cancer. A cell-based imaging assay that monitored the translocation of the Akt effector protein, Forkhead box O (FOXO), from the cytoplasm to the nucleus was employed to screen a collection of 33,992 small molecules. The positive compounds were used to screen kinases known to be involved in FOXO translocation. Pyrazolopyrimidine derivatives were found to be potent FOXO relocators as well as biochemical inhibitors of PI3Kα. A combination of virtual screening and molecular modeling led to the development of a structure-activity relationship, which indicated the preferred substituents on the pyrazolopyrimidine scaffold. This leads to the synthesis of ETP-45658, which is a potent and selective inhibitor of phosphoinositide 3-kinases and demonstrates mechanism of action in tumor cell lines and in vivo in treated mice.