Shirley K. Knauer

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.

A phosphorylation-acetylation switch regulates STAT1 signaling

Cytokines such as interferons (IFNs) activate signal transducers and activators of transcription (STATs) via phosphorylation. Histone deacetylases (HDACs) and the histone acetyltransferase (HAT) CBP dynamically regulate STAT1 acetylation. Here we show that acetylation of STAT1 counteracts IFN-induced STAT1 phosphorylation, nuclear translocation, DNA binding, and target gene expression. Biochemical and genetic experiments altering the HAT/HDAC activity ratio and STAT1 mutants reveal that a phospho-acetyl switch regulates STAT1 signaling via CBP, HDAC3, and the T-cell protein tyrosine phosphatase (TCP45). Strikingly, inhibition of STAT1 signaling via CBP-mediated acetylation is distinct from the functions of this HAT in transcriptional activation. STAT1 acetylation induces binding of TCP45, which catalyzes dephosphorylation and latency of STAT1. Our results provide a deeper understanding of the modulation of STAT1 activity. These findings reveal a new layer of physiologically relevant STAT1 regulation and suggest that a previously unidentified balance between phosphorylation and acetylation affects cytokine signaling.

Nuclear and Cytoplasmic Survivin: Molecular Mechanism, Prognostic, and Therapeutic Potential

Survivins proposed dual role as an apoptosis inhibitor and a mitotic effector positioned it in the front line of cancer research. Notably, survivin is detected as a cytoplasmic and nuclear protein in cancer patients, which stimulated numerous studies to investigate and to speculate on the functional and prognostic significance of its dynamic localization. Recent evidence shows that the direct interaction of survivin with the nuclear export receptor Crm1 is critically involved in its intracellular localization and cancer-relevant functions. Here, we review our current understanding of the Crm1/survivin interface and discuss its potential prognostic and therapeutic relevance.

Nuclear export is essential for the tumor-promoting activity of survivin

Survivin appears to function as an apoptosis inhibitor and a regulator of cell division during development and tumorigenesis. Here we report the molecular characterization of the nucleocytoplasmic transport of survivin and its potential implications for tumorigenesis. We identified an evolutionary conserved Crm1‐dependent nuclear export signal (NES) in survivin. In dividing cells, the NES is essential for tethering survivin and the survivin/Aurora‐B kinase complex to the mitotic machinery, which in turn appears to be essential for proper cell division. In addition, export seems to be required for the cytoprotective activity of survivin, as export‐deficient survivin fails to protect tumor cells against chemo‐and radiotherapy‐induced apoptosis. These findings appear to be clinically relevant since preferential nuclear localization of survivin correlated with enhanced survival in colorectal cancer patients. Targeting survivins nuclear export by the application of NES‐specific antibodies promoted its nuclear accumulation and inhibited its cytoprotective function. We demonstrate that nuclear export is essential for the biological activity of survivin and promote the identification of molecular decoys to specifically interfere with survivins nuclear export as potential anticancer therapeutics. Knauer, S. K., Krämer, O. H., Knösel, T., Engels, K., Rödel, F., Kovács, A. F., Dietmaier, W., Klein‐Hitpass, L., Habtemichael, N., Schweitzer, A., Brieger, J., Rödel, C., Mann, W., Petersen, I., Heinzel, T., Stauber, R. H. Nuclear export is essential for the tumor‐promoting activity of survivin. FASEB J. 21, 207–216 (2007)

The Survivin–Crm1 interaction is essential for chromosomal passenger complex localization and function

The chromosomal passenger complex (CPC) of Aurora‐B, Borealin, INCENP (inner centromere protein) and Survivin coordinates essential chromosomal and cytoskeletal events during mitosis. Here, we show that the nuclear export receptor Crm1 is crucially involved in tethering the CPC to the centromere by interacting with a leucine‐rich nuclear export signal (NES), evolutionarily conserved in all mammalian Survivin proteins. We show that inhibition of the Survivin–Crm1 interaction by treatment with leptomycin B or by RNA‐interference‐mediated Crm1 depletion prevents centromeric targeting of Survivin. The genetic inactivation of the Survivin–Crm1 interaction by mutation of the NES affects the correct localization and function of Survivin and the CPC during mitosis. By contrast, CPC assembly does not seem to require the Survivin–Crm1 interaction. Our report shows the functional significance of the Survivin–Crm1 interface and provides a novel link between the mitotic effector Crm1 and the CPC.

Dynamic intracellular survivin in oral squamous cell carcinoma : underlying molecular mechanism and potential as an early prognostic marker

Survivin functions as an apoptosis inhibitor and a regulator of cell division in many tumours. The intracellular localization of survivin in tumours has been suggested as a prognostic marker. However, current reports are inconsistent and the underlying molecular mechanisms are not understood. The present study has examined the localization and prognostic value of nuclear and cytoplasmic survivin in the pre‐therapeutic biopsies from 71 oral and oropharyngeal squamous carcinoma (OSCC) patients. Statistical analysis indicated that preferential nuclear versus cytoplasmic survivin correlated with favourable versus unfavourable disease outcome. Uni‐ and multi‐variate analysis showed that in contrast to total survivin expression, the difference between nuclear and cytoplasmic survivin was a strong predictor for relapse‐free survival (p = 0.0003). As a potential underlying molecular mechanism, it is shown in OSCC cell lines that predominantly cytoplasmic survivin mediates protection against chemo‐ and radio‐therapy‐induced apoptosis. Importantly, the cytoplasmic localization of survivin is regulated by its nuclear export signal (NES), and export‐deficient nuclear survivin is not cytoprotective. This study suggests that the difference between cytoplasmic and nuclear survivin is an indicator for survivin activity in tumour cells. Thus, this difference may serve as a predictive marker of outcome in OSCC patients undergoing multi‐modality therapy. The pharmacogenetic interference with survivins cytoplasmic localization is also to be pursued as a potential therapeutic strategy. Copyright

The role of survivin for radiation therapy. Prognostic and predictive factor and therapeutic target.

Background:Survivin, the smallest member of the inhibitor of apoptosis protein (IAP) family, is a bifunctional protein that has been implicated in both control of cell division and inhibition of apoptosis.Material and Methods:This review specially focuses on clinical and experimental data on the relevance of survivin in radiooncology and its role as a therapeutic target to radiosensitize tumor cells.Results:As compared to normal tissue, survivin is overexpressed in tumors and appears to be closely related to tumor malignancy and treatment response. In addition, survivin is involved in the resistance of tumor cells to both chemotherapy and ionising irradiation. Due to these properties, survivin has been proposed as an attractive target for anticancer therapies. Several preclinical studies have demonstrated that suppression of survivin, by the use of antisense oligonucleotides, small interfering RNAs, ribozymes and the application of dominant negative mutants, increases apoptotosis, diminishes tumor cell survival and reduces tumor growth potential.Conclusion:Survivin displays a relevant prognostic and predictive factor as well as a promising molecular target to improve the effectiveness of radiotherapy.Hintergrund:Survivin, das kleinste Mitglied der „Inhibitor of Apoptosis Protein“ (IAP)-Familie, ist ein bifunktionelles Protein mit essentiellen Funktionen in der Zellteilung und Hemmung der Apoptose.Material und Methoden:Dieser Artikel konzentriert sich auf klinische und experimentelle Daten zur Relevanz von Survivin in der Radioonkologie sowie dessen Rolle als therapeutische Zielstruktur für eine Radiosensibilisierung von Tumorzellen.Ergebnisse:Im Vergleich zu Normalgeweben wird Survivin in Tumoren überexprimiert und ist eng mit der Tumormalignität und dem Therapieansprechen assoziiert. Zudem ist Survivin an der Vermittlung einer Tumorzellresistenz gegenüber Chemotherapie und ionisierender Bestrahlung beteiligt. Aufgrund dieser Eigenschaften wird Survivin als eine attraktive Zielstruktur für die Krebstherapie angesehen. In mehreren präklinischen Untersuchungen konnte gezeigt werden, dass durch eine Antisense-Oligonukleotid-, siRNA- oder Ribozym-vermittelte Hemmung sowie nach Applikation dominant negativer Mutanten die Apoptoserate gesteigert wird. Die Überlebensrate der Krebszellen wie auch das Tumorwachstumspotential können dadurch gemindert werden.Schlussfolgerung:Survivin zeigt hohe Relevanz als prognostischer und prädiktiver Marker und stellt einen vielversprechenden molekularen Angriffspunkt zur Verbesserung der Effektivität der Radiotherapie dar.

The Survivin Isoform Survivin-3B is Cytoprotective and can Function as a Chromosomal Passenger Complex Protein

Survivin is described as a bifunctional protein inhibiting apoptosis and regulating mitosis. However, the biological functions and contributions to cancer progression of survivin splicevariants are controversially discussed. We here show that the intracellular localization of 5 these splice variants depends on a Crm1-dependent nuclear export signal (NES) present in survivin, survivin-2B and survivin-3B, but absent in survivin-ΔEx3 and survivin-2α. Survivin isoforms lack an active nuclear import signal and are able to enter the nucleus by passive diffusion. Only survivin-3B but none of the other splice variants is cytoprotective and able to efficiently interact with chromosomal passenger complex (CPC) proteins. The NES together 10 with efficient CPC formation is required for the cytoprotective activity of survivin isoforms, aswell as for their correct localization and function during cell division. In the tumours from breast, colorectal, head and neck cancer, lymphoma and leukemia patients, survivin and survivin-2B were found overexpressed. However, survivin was the predominant form detected, and the other survivin isoforms were only expressed at low levels in tumours. Our data 15 provide a molecular rationale for the localization and activity of survivin variants, and conclude that survivin isoforms are unlikely to modulate survivin in trans in cancer patients.

The Role of Survivin for Radiation Therapy

Background:Survivin, the smallest member of the inhibitor of apoptosis protein (IAP) family, is a bifunctional protein that has been implicated in both control of cell division and inhibition of apoptosis.Material and Methods:This review specially focuses on clinical and experimental data on the relevance of survivin in radiooncology and its role as a therapeutic target to radiosensitize tumor cells.Results:As compared to normal tissue, survivin is overexpressed in tumors and appears to be closely related to tumor malignancy and treatment response. In addition, survivin is involved in the resistance of tumor cells to both chemotherapy and ionising irradiation. Due to these properties, survivin has been proposed as an attractive target for anticancer therapies. Several preclinical studies have demonstrated that suppression of survivin, by the use of antisense oligonucleotides, small interfering RNAs, ribozymes and the application of dominant negative mutants, increases apoptotosis, diminishes tumor cell survival and reduces tumor growth potential.Conclusion:Survivin displays a relevant prognostic and predictive factor as well as a promising molecular target to improve the effectiveness of radiotherapy.Hintergrund:Survivin, das kleinste Mitglied der „Inhibitor of Apoptosis Protein“ (IAP)-Familie, ist ein bifunktionelles Protein mit essentiellen Funktionen in der Zellteilung und Hemmung der Apoptose.Material und Methoden:Dieser Artikel konzentriert sich auf klinische und experimentelle Daten zur Relevanz von Survivin in der Radioonkologie sowie dessen Rolle als therapeutische Zielstruktur für eine Radiosensibilisierung von Tumorzellen.Ergebnisse:Im Vergleich zu Normalgeweben wird Survivin in Tumoren überexprimiert und ist eng mit der Tumormalignität und dem Therapieansprechen assoziiert. Zudem ist Survivin an der Vermittlung einer Tumorzellresistenz gegenüber Chemotherapie und ionisierender Bestrahlung beteiligt. Aufgrund dieser Eigenschaften wird Survivin als eine attraktive Zielstruktur für die Krebstherapie angesehen. In mehreren präklinischen Untersuchungen konnte gezeigt werden, dass durch eine Antisense-Oligonukleotid-, siRNA- oder Ribozym-vermittelte Hemmung sowie nach Applikation dominant negativer Mutanten die Apoptoserate gesteigert wird. Die Überlebensrate der Krebszellen wie auch das Tumorwachstumspotential können dadurch gemindert werden.Schlussfolgerung:Survivin zeigt hohe Relevanz als prognostischer und prädiktiver Marker und stellt einen vielversprechenden molekularen Angriffspunkt zur Verbesserung der Effektivität der Radiotherapie dar.

Histone deacetylase inhibitors and hydroxyurea modulate the cell cycle and cooperatively induce apoptosis

Therapy resistance represents a major problem for disease management in oncology. Histone deacetylase inhibitors (HDACi) have been shown to modulate the cell cycle, to induce apoptosis and to sensitize cancer cells for other chemotherapeutics. Our study shows that the HDACi valproic acid (VPA) and the ribonucleotide reductase inhibitor hydroxyurea (HU) potentiate the pro-apoptotic effects of each other towards several cancer cell lines. This correlates with the HU-induced degradation of the cyclin-dependent kinase inhibitors (CDKI) p21 and p27, mediated by the proteasome or caspase-3. Moreover, we found that caspase-3 activation is required for VPA-induced apoptosis. Remarkably, p21 and p27 can confer resistance against VPA and HU. Both CDKI interact with caspase-3 and compete with other caspase-3 substrates. Hence, p21 and p27 may contribute to chemotherapy resistance as apoptosis inhibitors. Since the biological effects of VPA and HU could be achieved at concentrations used in current treatment protocols, the combined application of these compounds might be considered as a potential strategy for cancer treatment.