Verena Fetz

Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology

In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.

NO Signaling Confers Cytoprotectivity through the Survivin Network in Ovarian Carcinomas

Despite considerable success in the treatment of epithelial ovarian cancer (EOC), therapy resistance counteracts improvement of long-term survival. The dual role of survivin as an apoptosis inhibitor and mitotic regulator has been associated with disease outcome. However, the molecular mechanisms involved in the deregulated expression in EOC of survivin need further investigation. Here, we show that high amounts of the nitric oxide (NO) donors, S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) or strong overexpression of the inducible nitric oxide synthase (iNOS) suppressed survivin levels via the p38MAPK pathway and triggered apoptosis in ovarian cancer cell lines (OCC). Importantly, low NO concentrations conferred resistance against carboplatin/paclitaxel-induced apoptosis. Cytoprotection was mediated by survivin because we observed its up-regulation subsequent to low SNAP/SNP doses or ectopic expression of low amounts of iNOS. Also, RNAi-mediated depletion of survivin blocked the antiapoptotic effects of NO signaling. Induction of survivin involves activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, which was antagonized by the PI3K-inhibitor, LY294002. Interestingly, application of the iNOS-inhibitor 1400W together with RNAi-mediated survivin down-regulation cooperatively enhanced drug-induced apoptosis in OCCs. The iNOS/survivin interdependencies seem to be also of clinical relevance because immunohistochemistry revealed that low iNOS levels correlate with survivin expression (P < 0.01) in carboplatin/paclitaxel-treated EOC patients with minimal postoperative residual tumor (n = 54). Also, iNOS and survivin expression were associated with increased risk for disease progression. Our study uncovers a novel molecular mechanism of how NO signaling may contribute to therapy resistance in EOC by modulating survivin expression. Pharmacogenetic iNOS/survivin-targeting strategies may hence be pursued to complement current treatment modalities in EOC.

Inducible NO synthase confers chemoresistance in head and neck cancer by modulating survivin

The dual role of the inducible NO synthase (iNOS) and NO signaling in head and neck squamous cell carcinoma (HNSCC) is a complex and can both promote or inhibit tumor progression. However, the underlying molecular mechanisms are not yet resolved in detail. We show for the first time that conditions, favoring low NO levels conferred resistance against cisplatin/taxol‐induced apoptosis in HNSCC cell lines. Cytoprotection was mediated by survivin, because we observed its upregulation subsequent to low doses of the NO donors S‐nitroso‐N‐acetyl‐penicillamine (SNAP) and sodium nitroprusside (SNP) or ectopic expression of physiologic amounts of iNOS. Also, RNAi‐mediated depletion of survivin blocked NOs anti‐apoptotic effects. Induction of survivin involves activation of the phosphatidylinositol‐3‐kinase/Akt (PI3K/Akt) pathway, which was antagonized by the PI3K‐inhibitor LY294002. Importantly, application of the iNOS‐specific inhibitor 1400W combined with RNAi‐mediated downregulation of survivin cooperatively enhanced drug‐induced cell death. The iNOS/survivin‐axis appears to be also of clinical relevance since immunohistochemistry revealed that iNOS expression correlated with enhanced survivin levels in HNSCC specimens. In contrast, high NO concentrations suppressed survivin levels in HNSCC but also in non‐malignant cells resulting in apoptosis. Cell death induced by high amounts of SNAP/SNP or by strong overexpression of iNOS involved activation of p38MAP‐kinase, which was counteracted by the p38MAP‐kinase inhibitor SB202190. Here, we provide evidence for a novel molecular mechanism how NO signaling may contribute to therapy resistance in HNSCC by modulating survivin expression. Our data further suggest pursuing pharmacogenetic iNOS/survivin‐targeting approaches as potential therapeutic strategies in head and neck cancer.

Dynamic survivin in head and neck cancer: Molecular mechanism and therapeutic potential

Although disease management of head and neck squamous cell carcinomas (HNSCC) has improved significantly, therapy resistance leading to tumor recurrence still counteracts improvement of long‐term survival. Consequently, identification of molecular markers that signal increased risk of treatment failure or, which can be exploited by targeted therapy, is urgently needed. Survivin is strongly expressed in HNSCC, and its proposed dual role as an apoptosis inhibitor and a mitotic effector positioned survivin in the front line of cancer research. Notably, survivin is detected as a cytoplasmic and as a nuclear protein in HNSCC patients, which stimulated numerous studies to investigate and to speculate on the functional and prognostic significance of its dynamic localization. This review focuses on our current understanding of the molecular mechanisms regulating survivins intracellular localization and discusses its potential prognostic and therapeutic relevance for head and neck cancer.

Translocation Biosensors – Cellular System Integrators to Dissect CRM1-Dependent Nuclear Export by Chemicogenomics

Fluorescent protein biosensors are powerful cellular systems biology tools for dissecting the complexity of cellular processes with high spatial and temporal resolution. As regulated nucleo-cytoplasmic transport is crucial for the modulation of numerous (patho)physiological cellular responses, a detailed understanding of its molecular mechanism would open up novel options for a rational manipulation of the cell. In contrast to genetic approaches, we here established and employed high-content cellular translocation biosensors applicable for dissecting nuclear export by chemicogenomics. A431 cell lines, stably expressing a translocation biosensor composed of glutathione S-transferase, GFP and a rational combination of nuclear import and export signals, were engineered by antibiotic selection and flow cytometry sorting. Using an optimized nuclear translocation algorithm, the translocation response could be robustly quantified on the Cellomics Arrayscan® VTI platform. Subsequent to assay optimization, the assay was developed into a higher density 384-well format high-content assay and employed for the screening of the 17K ChemBioNet compound collection. This library was selected on the basis of a genetic algorithm used to identify maximum common chemical substructures in a database of annotated bioactive molecules and hence, is well-placed in the chemical space covered by bioactive compounds. Automated multiparameter data analysis combined with visual inspection allowed us to identify and to rationally discriminate true export inhibitors from false positives, which included fluorescent compounds or cytotoxic substances that dramatically affected the cellular morphology. A total of 120 potential hit compounds were selected for Cellomics Arrayscan® VTI based rescreening. The export inhibitory activity of 20 compounds effective at concentrations < 25 μM were confirmed by fluorescence microscopy in several cell lines. Interestingly, kinetic analysis allowed the identification of inhibitors capable to interfere with the export receptor CRM1-mediated nuclear export not only in an irreversible, but also in a reversible fashion. In sum, exploitation of biosensor based screening allows the identification of chemicogenomic tools applicable for dissecting nucleo-cytoplasmic transport in living cells.

Chemico-genetic strategies to inhibit the leukemic potential of threonine aspartase-1.

Chromosomal rearrangements of the mixed lineage leukemia (MLL) gene with numerous partner genes are frequently found in acute myeloid (AML) and acute lymphoblastic leukemia (ALL). Although the pathomechanism of t(4;11)-mediated leukemia is still discussed, expression of the AF4•MLL fusion enhanced the repopulating potential of CD34+-cells, and led to the development of predominantly proB-ALL in a mouse model. The AF4•MLL protein contains cleavage-sites for Threonine Aspartase-1 (Taspase1). Upon processing by Taspase1, the AF4•MLL cleavage products form a protein complex resistant to SIAH-mediated degradation and activate oncogenic programs. Furthermore, Taspase1 is overexpressed in liquid and solid human cancers suggesting that Taspase1 is coopted to promote and sustain tumorigenesis. As genetic deletion of Taspase1 in the mouse produced no overt deficiencies, inhibition of Taspase1 may offer novel anticancer strategies, including the treatment of leukemias. Human Taspase1 encodes a protease of 420 amino acids (aa) cleaving substrates in trans by recognizing a conserved peptide motif (Q3[F,I,L,V]2D1↓G1’x2’D3’D4’). Unfortunately, Taspase1’s activity is not affected by common protease inhibitors, currently precluding the assessment of its clinical and therapeutic relevance. Here, we present our endeavors to target Taspase1’s oncogenic potential by (i) overexpression of inactive Taspase1 variants, and (ii) testing a putative Taspase1 inhibitor.

Functional Characterization of Novel Mutations Affecting Survivin (BIRC5)‐Mediated Therapy Resistance in Head and Neck Cancer Patients

Survivin (BIRC5) is an acknowledged cancer therapy‐resistance factor and overexpressed in head and neck squamous cell carcinomas (HNSCC). Driven by its nuclear export signal (NES), Survivin shuttles between the nucleus and the cytoplasm, and is detectable in both cellular compartments in tumor biopsies. Although predominantly nuclear Survivin is considered a favorable prognostic disease marker for HNSCC patients, the underlying molecular mechanisms are not resolved. Hence, we performed immunohistochemical and mutational analyses using laser capture microdissection on HNSCC biopsies from patients displaying high levels of nuclear Survivin. We found somatic BIRC5 mutations, c.278T>C (p.Phe93Ser), c.292C>T (p.Leu98Phe), and c.288A>G (silent), in tumor cells, but not in corresponding normal tissues. Comprehensive functional characterization of the Survivin mutants by ectopic expression and microinjection experiments revealed that p.Phe93Ser, but not p.Leu98Phe inactivated Survivins NES, resulted in a predominantly nuclear protein, and attenuated Survivins dual cytoprotective activity against chemoradiation‐induced apoptosis. Notably, in xenotransplantation studies, HNSCC cells containing the p.Phe93Ser mutation responded significantly better to cisplatin‐based chemotherapy. Collectively, our results underline the disease relevance of Survivins nucleocytoplasmic transport, and provide first evidence that genetic inactivation of Survivins NES may account for predominantly nuclear Survivin and increased therapy response in cancer patients.

Arginine residues within the DNA binding domain of STAT3 promote intracellular shuttling and phosphorylation of STAT3

Acetylation-dependent inactivation of STAT1 can be mimicked by the exchange of its lysine residues K410 and K413 to glutamine residues. STAT3 harbors non-acetylatable arginine moieties at the corresponding sites R414 and R417. It is unclear whether the mutation of these sites to glutamine residues antagonizes STAT3 activation. Here, we show that an arginine-glutamine-exchange at the STAT3 moieties R414 and R417 (R414Q and R417Q) reduces cytokine-dependent tyrosine phosphorylation of STAT3. This inhibitory effect can be partially rescued by phosphatase inhibition. In addition, the R414Q and R417Q mutations enhance the nuclear accumulation of unphosphorylated STAT3. STAT3 R414Q and STAT3 R417Q show a reduced response to cytokine stimulation emanating from the plasma membrane. Moreover, these STAT3 mutants have no direct inhibitory effect on the cytokine-induced activation of STAT1/STAT3-mediated gene expression. Since the mutations R414Q and R417Q reside within the STAT3 DNA binding domain (DBD), the STAT3 R414Q and R417Q mutants also lack intrinsic activity as transcription factors. Furthermore, in contrast to wild-type STAT3 they cannot compensate for a loss of STAT1 and they cannot promote STAT1/STAT3-dependent transcriptional activation. We further analyzed a STAT3 arginine-lysine-exchange mutant (R414K/R417K). This molecule mimics corresponding lysine residues found within the DBD of STAT1. Compared to wild-type STAT3, the STAT3 R414K/R417K mutant shows attenuated tyrosine phosphorylation and it is a less active transcription factor. In addition, STAT3 R414K/R417K is not activated by deacetylase inhibition. On the other hand, C-terminal acetylation of STAT3 is intact in STAT3 R414K/R417K. Our results suggest that the exchange of amino acid residues within the DBDs of STAT1/STAT3 affects their phosphorylation as well as their intracellular shuttling.

Translocation Biosensors—Versatile Tools to Probe Protein Functions in Living Cells

In this chapter, you will learn how to use translocation biosensors to investigate protein functions in living cells. We here present three classes of modular protein translocation biosensors tailored to investigate: (1) signal-mediated nucleo-cytoplasmic transport, (2) protease activity, and (3) protein-protein interactions. Besides the mapping of protein function, the biosensors are also applicable to identify chemicals and/or (nano) materials modulating the respective protein activities and can also be exploited for RNAi-mediated genetic screens.

TFIIA transcriptional activity is controlled by a ‘cleave-and-run’ Exportin-1/Taspase 1-switch

Transcription factor TFIIA is controlled by complex regulatory networks including proteolysis by the protease Taspase 1, though the full impact of cleavage remains elusive. Here, we demonstrate that in contrast to the general assumption, de novo produced TFIIA is rapidly confined to the cytoplasm via an evolutionary conserved nuclear export signal (NES, amino acids 21VINDVRDIFL30), interacting with the nuclear export receptor Exportin-1/chromosomal region maintenance 1 (Crm1). Chemical export inhibition or genetic inactivation of the NES not only promotes TFIIAs nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIAs nuclear accumulation by NES masking, and modulates its transcriptional activity. Moreover, TFIIA complex formation with the TATA box binding protein (TBP) is cooperatively enhanced by inhibition of proteolysis and nuclear export, leading to an increase of the cell cycle inhibitor p16INK, which is counteracted by prevention of TBP binding. We here identified a novel mechanism how proteolysis and nuclear transport cooperatively fine-tune transcriptional programs.