Corina Borghouts

The function of Stat3 in tumor cells and their microenvironment.

Stat3 was initially recognized as a transcription factor and mediates the nuclear action of many different cytokines and growth factors. In addition to its roles in normal cell function, the inappropriate activation of Stat3 in tumor cells has attracted the attention of tumor biologists and has led to the consideration of Stat3 as a drug target. The induction of Stat3 activity under physiological circumstances is transient and many different levels of activation and deactivation have been defined. In addition to kinases and phosphatases, the SOCS proteins and the PIAS proteins have been recognized as negatively regulating components, which fine-tune the extent and the duration of Stat3 function. Its nuclear cytoplasmic shuttling is exquisitely regulated and adds to the complexity of Stat3 action. Newly discovered associations with cytoplasmic molecules suggest functions outside the conventional transcriptional regulation context. High molecular weight transcription complexes suggest that Stat3 might assume roles in transcriptional induction as well as in transcriptional suppression. The aberrant activation in tumor cells and the central function of Stat3 in the communication between cells of the immune system and tumor cells are of great interest for translational research projects and innovative drug development.

Copper-Modulated Gene Expression and Senescence in the Filamentous Fungus Podospora anserina

ABSTRACT We have previously shown that the control of cellular copper homeostasis by the copper-modulated transcription factor GRISEA has an important impact on the phenotype and lifespan of Podospora anserina. Here we demonstrate that copper depletion leads to the induction of an alternative respiratory pathway and to an increase in lifespan. This response compensates mitochondrial dysfunctions via the expression of PaAox, a nuclear gene coding for an alternative oxidase. It resembles the retrograde response inSaccharomyces cerevisiae. In P. anserina, this pathway appears to be induced by specific impairments of the copper-dependent cytochrome c oxidase. It is not induced as the result of a general decline of mitochondrial functions during senescence. We cloned and characterized PaAox. Transcript levels are decreased when cellular copper, superoxide, and hydrogen peroxide levels are raised. Copper also controls transcript levels ofPaSod2, the gene encoding the mitochondrial manganese superoxide dismutase (PaSOD2). PaSod2 is a target of transcription factor GRISEA. During the senescence of wild-type strain s, the activity of PaSOD2 decreases, whereas the activity of the cytoplasmic copper/zinc superoxide dismutase (PaSOD1) increases. Collectively, the data explain the postponed senescence of mutant grisea as a defined consequence of copper depletion, ultimately leading to a reduction of oxidative stress. Moreover, they suggest that during senescence of the wild-type strain, copper is released from mitochondria. The involved mechanism is unknown. However, it is striking that the permeability of mitochondrial membranes in animal systems changes during apoptosis and that mitochondrial proteins with an important impact on this type of cellular death are released.

The Biological Functions of the Versatile Transcription Factors STAT3 and STAT5 and New Strategies for their Targeted Inhibition

Signal transducers and activators of transcription (STATs) comprise a unique family of transcription factors, which transmit the interactions of cytokines, hormones and growth factors with their cell surface receptors into transcriptional programs. The mechanism of STAT activation has been well-established and comprises tyrosine phosphorylation, dimerization, nuclear translocation, binding to specific DNA response elements, recruitment of co-activators or co-repressors and transcriptional induction or repression of target genes. Gene deletion, microarrays, proteomics and chromatin immunoprecipitation experiments have revealed target genes with a broad range of functions regulated by STAT3 and STAT5. In the mammary gland, STAT5-induced genes contribute mainly to the prolactin dependent lobulo-alveolar development, whereas STAT3 induced genes control apoptosis during involution. Crucial effects have also been observed in other tissues. The germ line deletion of STAT3 or STAT5 causes early embryonal or perinatal lethality in mice. STAT5 is also required for proliferation of T- and B-cells and hematopoietic stem cell self-renewal. Deregulated STAT activity is often found associated with tumorigenesis and activated STATs seem to be limiting components in tumor cells. This review summarizes the functions of STAT3 and STAT5 in different cell types and the strategies that are used to counteract their action in tumor cells.

Copper homeostasis and aging in the fungal model system Podospora anserina: differential expression of PaCtr3 encoding a copper transporter.

Lifespan extension of Podospora anserina mutant grisea is caused by a loss-of-function mutation in the nuclear gene Grisea. This gene encodes the copper regulated transcription factor GRISEA recently shown to be involved in the expression of PaSod2 encoding the mitochondrial manganese superoxide dismutase. Here we report the identification and characterization of a second target gene. This gene, PaCtr3, encodes a functional homologue of the Saccharomyces cerevisiae high affinity copper permease yCTR3. PaCtr3 is not expressed in the grisea mutant confirming the assumption that the extension of lifespan is primarily caused by cellular copper limitation and a switch from a cytochrome oxidase (COX)-dependent to and alternative oxidase (AOX)-dependent respiration. Transcript levels of PaCtr3 and PaSod2 respond to copper, iron, manganese and zinc. Transcription of PaCtr3 was found to be down-regulated during senescence of wild-type cultures suggesting that the intracellular copper concentration is raised in old cultures. A two hybrid analysis suggested that GRISEA acts as a homodimer. In accordance, an inverted repeat was identified as a putative binding sequence in the promoter region of PaCtr3 and of PaSod2. Finally, the expression of PaCtr3 in transformants of the grisea mutant led to lifespan shortening. This effect correlates with the activity of the copper-dependent COX demonstrating a strong link between copper-uptake, respiration and lifespan.

Peptide aptamers: recent developments for cancer therapy

During the past two decades, our understanding of oncogenesis has advanced considerably and many new signalling pathways have been identified. Differences in signalling events that distinguish normal cells from tumour cells provide new targets for the development of anticancer agents. Peptide aptamers are small peptide sequences that have been selected to recognise a predetermined target protein domain and are potentially able to interfere with its function. They represent useful molecules for manipulating protein function in vivo. The isolation and use of specific peptide aptamers as inhibitors of individual signalling components, essential in cancer development and progression, provides a new challenge for drug development. Although peptides make up only a small fraction of current therapeutics, their potential is being enhanced by new developments affecting their modification, stability, delivery and their successful application in preclinical settings. This review summarises the methods that can be used for the isolation and delivery of peptide aptamers, as well as the important achievements that have been made using such peptide aptamers in different systems. The applicability of peptide aptamers as novel cancer therapeutics will be discussed.

Respiration, copper availability and SOD activity in P. anserina strains with different lifespan.

P. anserina mutants with impairments in complex IV (COX) of the respiratory chain are characterized by an increase in lifespan. Examples are the nuclear grisea mutant with a moderate lifespan extension (60%) and the immortal extranuclear ex1 mutant. Here we report data demonstrating that in mutant ex1 the level of the alternative oxidase (PaAOX) is significantly higher than in mutant grisea. PaAOX levels appear to be reversely dependent on COX activity. The activity profile of superoxide dismutases in the ex1 mutant resembles the profile in senescent wild-type cultures with a high cytoplasmic copper/zinc superoxide dismutase (PaSOD1) and a low mitochondrial manganese superoxide dismutase (PaSOD2) activity. In the grisea mutant, PaSOD1 activity is only detectable in cultures grown in copper-supplemented medium. The two copper-regulated genes PaCtr3 (coding for a high affinity copper transporter) and PaSod2 are not expressed in the two mutants grown in standard medium. The repression of these genes as well as the activity of PaSOD1 is dependent on the availability of cellular copper, which appears to be high in COX-deficient strains such as mutant ex1 and in the senescent wild-type strain. In the wild-type, changes in the cellular localization of copper and in the delivery of this metal to different proteins appear to occur during senescence. Collectively, the data explain the characteristic lifespan of the investigated strains as the result of differences in energy transduction and in the machinery protecting against oxidative stress.

Monomeric Recombinant Peptide Aptamers Are Required for Efficient Intracellular Uptake and Target Inhibition

Signal transduction events often involve the assembly of protein complexes dependent on modular interactions. The inappropriate assembly of modular components plays a role in oncogenic transformation and can be exploited for therapeutic purposes. Selected peptides embedded in the context of a scaffold protein can serve as competitive inhibitors of intracellular protein functions in cancer cells. Therapeutic application depends on binding specificities and affinities, as well as on the production and purification characteristics of the peptide aptamers and their delivery into cells. We carried out experiments to improve the properties of the scaffold. We found that the commonly used bacterial thioredoxin scaffold is suboptimal for therapeutic purposes because it aggregates during purification and is most likely immunogenic in humans. We compared the properties of peptide aptamers embedded in three alternative scaffold structures: a coiled-coil stem-loop structure, a dimerization domain, and human thioredoxin (hTrx). We found that only the hTrx molecule can be efficiently produced in bacteria and purified with high yield. We removed five internal cysteines of hTrx to circumvent aggregation during purification, which is a prerequisite for efficient transduction. Insertion of our previously characterized peptide aptamers [e.g., specifically binding signal transducer and activator of transcription 3 (Stat3)] into the modified hTrx scaffold retained their target binding properties. Addition of a protein transduction domain, consisting of nine arginines, results in a fusion protein, which is taken up by cultured cells. We show that treatment of glioblastoma cells, expressing constitutively activated Stat3, with the purified peptide aptamers strongly inhibits Stat3 signaling, causing cell growth arrest and inducing apoptosis. (Mol Cancer Res 2008;6(2):267–81)

Peptide Aptamers with Binding Specificity for the Intracellular Domain of the ErbB2 Receptor Interfere with AKT Signaling and Sensitize Breast Cancer Cells to Taxol

The ErbB2 receptor tyrosine kinase is overexpressed in ∼30% of breast tumor cases and its overexpression correlates with an unfavorable prognosis. A major contributor for this course of the disease is the insensitivity of these tumors toward chemotherapy. Monoclonal antibodies, inhibiting the ligand-induced activation of the receptor and tyrosine kinase inhibitors acting on the intrinsic enzymatic activity of the intracellular domain, have been developed as targeted drugs. Both have been shown to be beneficial for breast cancer patients. We targeted a third aspect of receptor function: its association with intracellular signaling components. For this purpose, we selected peptide aptamers, which specifically interact with defined domains of the intracellular part of the receptor. The peptide aptamers were selected from a random peptide library using a yeast two-hybrid system with the intracellular tyrosine kinase domain of ErbB2 as a bait construct. The peptide aptamer AII-7 interacts with high specificity with the ErbB2 receptor in vitro and in vivo. The aptamers colocalized with the intracellular domain of ErbB2 within cells. We investigated the functional consequences of the aptamer interaction with the ErbB2 receptor within tumor cells. The aptamer sequences were either expressed intracellularly or introduced into the cells as recombinant aptamer proteins. The phosphorylation of p42/44 mitogen-activated protein kinase was nearly unaffected and the activation of signal transducers and activators of transcription-3 was only modestly reduced. In contrast, they strongly inhibited the induction of AKT kinase in MCF7 breast cancer cells treated with heregulin, whereas AKT activation downstream of insulin-like growth factor I or epidermal growth factor receptor was not or only slightly affected. High AKT activity is responsible for the enhanced resistance of ErbB2-overexpressing cancer cells toward chemotherapeutic agents. Peptide aptamer interference with AKT activation resulted in the restoration of regular sensitivity of breast cancer cells toward Taxol. (Mol Cancer Res 2006;4(12):983–98)

Peptide Aptamer Libraries

Peptide aptamers are molecules that bind to protein targets and are able to interfere with their functions. In the past, important achievements have been made using such peptide aptamers in different approaches and for various purposes. Peptide aptamers are comprised of a variable peptide region of 8 to 20 amino acids in length, which is displayed by a scaffold protein. An overview of the numerous scaffold proteins that have been investigated for their suitability to present peptide aptamers will be given. To identify peptide aptamers efficiently and specifically binding to a predetermined target, two eukaryotic systems have been used in multiple studies: a modified version of the Gal4 yeast-two-hybrid system and the optimized LexA interaction trap system. The two yeast systems are compared and the design of high-complexity peptide aptamer libraries for these systems is described. Although the yeast-two-hybrid system is based on intracellular interactions mammalian screens, performed in cell culture experiments, are sometimes preferred or required. We will give an overview of the mammalian selection systems available, which are based on the expression of peptide aptamers in retroviral or lentiviral vectors. We will show that the isolation and use of peptide aptamers as inhibitors of individual signaling components represents a new challenge for drug development.

Copper-dependence of mitochondrial DNA rearrangements in Podospora anserina

Abstract Rearrangements of the mitochondrial DNA (mtDNA) are a hallmark of senescence in wild-type strains of the ascomycete P. anserina. These rearrangements include the systematic amplification of the first intron (pl-intron) of the cytochrome oxidase subunit-I gene (CoI) as a circular DNA molecule (plDNA). In addition, deletions and amplifications of other regions of the mtDNA occur. The molecular basis of the underlying processes is not understood in detail. A comparative analysis of the wild-type strain and of the long-lived mutant grisea, affected in the uptake of copper, revealed that mtDNA instabilities are dependent on the availability of cellular copper. In the mutant, the first steps in the corresponding pathway, including the transcription of the CoI gene, the splicing of the pl-intron and the transposition of this mobile element, are not impaired. In contrast, recombination processes between short direct repeats, as well as rearrangements between two tandem intron copies leading to the formation of plDNA, appear to be affected. Additional copper in the growth medium rescues this molecular phenotype. We suggest that copper is a cofactor of a component of the molecular machinery leading to the characteristic age-related mtDNA rearrangements.