Carolin Bier

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 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.

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.

Nucleocytoplasmic Shuttling and the Biological Activity of Mouse Survivin are Regulated by an Active Nuclear Export Signal

Survivin appears to function as a regulator of cell division and as an apoptosis inhibitor in many species. Here, we characterized the nucleocytoplasmic transport of mouse survivin140, and its splice variants survivin121 and survivin40. We show that the dynamic intracellular localization of survivin140 is mediated by a Crm1‐dependent nuclear export signal (NES) present also in survivin121, but absent in survivin40. In contrast, neither survivin nor survivin splice variants contain an active nuclear import signal and seem to enter the nucleus by passive diffusion. The activity of the NES is required for survivin‐mediated protection against cell death inducing stimuli and influences protein degradation. During mitosis, NES‐deficient survivin variants fail to correctly localize to the mitotic machinery and promote proper cell division. In vivo and in vitro protein interaction assays show that survivin140 and survivin121 as well as their export‐deficient mutants are able to form homo‐ as well as heterodimers. The trans‐dominant negative phenotype observed upon expression of export‐deficient survivin appears, therefore, to be mediated by the formation of inactive survivin heterodimers. The survivin–Crm1 axis is essential for the biological activities of murine survivin, and mouse models will allow investigating its functional implications during development and tumorigenesis.

Cell-based Analysis of Structure-Function Activity of Threonine Aspartase 1

Taspase1 is a threonine protease responsible for cleaving intracellular substrates. As such, (de)regulated Taspase1 function is expected not only to be vital for ordered development but may also be relevant for disease. However, the full repertoires of Taspase1 targets as well as the exact biochemical requirements for its efficient and substrate-specific cleavage are not yet resolved. Also, no cellular assays for this protease are currently available, hampering the exploitation of the (patho)biological relevance of Taspase1. Here, we developed highly efficient cell-based translocation biosensor assays to probe Taspase1 trans-cleavage in vivo. These modular sensors harbor variations of Taspase1 cleavage sites and localize to the cytoplasm. Expression of Taspase1 but not of inactive Taspase1 mutants or of unrelated proteases triggers proteolytic cleavage and nuclear accumulation of the biosensors. Employing our assay combined with scanning mutagenesis, we identified the sequence and spatial requirements for efficient Taspase1 processing in liquid and solid tumor cell lines. Collectively, our results defined an improved Taspase1 consensus recognition sequence, Q3(F/I/L/V)2D1↓G1′X2′D3′D4′, allowing the first genome-wide bioinformatic identification of the human Taspase1 degradome. Among the 27 most likely Taspase1 targets are cytoplasmic but also nuclear proteins, such as the upstream stimulatory factor 2 (USF2) or the nuclear RNA export factors 2/5 (NXF2/5). Cleavage site recognition and proteolytic processing of selected targets were verified in the context of the biosensor and for the full-length proteins. We provide novel mechanistic insights into the function and bona fide targets of Taspase1 allowing for a focused investigation of the (patho)biological relevance of this type 2 asparaginase.

Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation

Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation.

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.

An otoprotective role for the apoptosis inhibitor protein survivin.

Hearing impairment caused by ototoxic insults, such as noise or gentamicin is a worldwide health problem. As the molecular circuitries involved are not yet resolved, current otoprotective therapies are rather empirical than rational. Here, immunohistochemistry and western blotting showed that the cytoprotective protein survivin is expressed in the human and guinea pig cochlea. In the guinea pig model, moderate noise exposure causing only a temporary hearing impairment transiently evoked survivin expression in the spiral ligament, nerve fibers and the organ of Corti. Mechanistically, survivin upregulation may involve nitric oxide (NO)-induced Akt signaling, as enhanced expression of the endothelial NO synthase and phosphorylated Akt were detectable in some surviving-positive cell types. In contrast, intratympanic gentamicin injection inducing cell damage and permanent hearing loss correlated with attenuated survivin levels in the cochlea. Subsequently, the protective activity of the human and the guinea pig survivin orthologs against the ototoxin gentamicin was demonstrated by ectopic overexpression and RNAi-mediated depletion studies in auditory cells in vitro. These data suggest that survivin represents an innate cytoprotective resistor against stress conditions in the auditory system. The pharmacogenetic modulation of survivin may thus provide the conceptual basis for the rational design of novel therapeutic otoprotective strategies.

The Importin‐Alpha/Nucleophosmin Switch Controls Taspase1 Protease Function

Taspase1 is a threonine protease suspected to process (patho)biologically relevant nuclear and cytoplasmic substrates, such as the mixed lineage leukemia protein. However, neither the mechanisms regulating Taspase1s intracellular localization nor their functional consequences are known. Analysis of endogenous and ectopically expressed Taspase1 detected the protease predominantly in the nucleus accumulating at the nucleolus. Microinjection and ectopic expression studies identified an evolutionarily conserved bipartite nuclear import signal (NLS) (amino acids 197KRNKRKLELA ERVDTDFMQLKKRR220) interacting with importin‐α. Notably, an NLS‐mutated, import‐deficient Taspase1 was biologically inactive. Although the NLS conferred nuclear transport already of the proenzyme, Taspase1s nucleolar localization required its autoproteolytic processing, triggering its interaction with the nucleolar shuttle protein nucleophosmin. In contrast, (auto)catalytically inactive Taspase1 mutants neither accumulated at the nucleolus nor bound nucleophosmin. Active nuclear import and interaction with nucleophosmin was found to be required for the formation of proteolytically active Taspase1 ensuring to efficiently process its nuclear targets. Intriguingly, coexpression of pathological nucleophosmin variants increased the amount of cytoplasmic Taspase1. Hence, Taspase1 appears to exploit the nuclear export activity of nucleophosmin to gain transient access to the cytoplasm required to also cleave its cytoplasmic substrates. Collectively, we here describe a hitherto unknown mechanism regulating the biological activity of this protease.