ABSTRACT
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 Survivin's NES, resulted in a predominantly nuclear protein, and attenuated Survivin's 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 Survivin's nucleocytoplasmic transport, and provide first evidence that genetic inactivation of Survivin's NES may account for predominantly nuclear Survivin and increased therapy response in cancer patients.
Subject(s)
Carcinoma, Squamous Cell/diagnosis , Head and Neck Neoplasms/diagnosis , Head and Neck Neoplasms/drug therapy , Inhibitor of Apoptosis Proteins/genetics , Active Transport, Cell Nucleus , Animals , Apoptosis/drug effects , Carcinoma, Squamous Cell/drug therapy , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cisplatin/therapeutic use , Cytoplasm/genetics , Cytoplasm/metabolism , Disease Models, Animal , Fatty Acids, Unsaturated/pharmacology , Female , Humans , Immunohistochemistry , Inhibitor of Apoptosis Proteins/metabolism , Mice , Mutation , Nuclear Export Signals/genetics , Prognosis , SurvivinABSTRACT
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.
Subject(s)
Blood Proteins/metabolism , Nanoparticles/chemistry , Blood Platelets/drug effects , Blood Platelets/metabolism , Cell Death/drug effects , Cell Line , Computational Biology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Humans , Microscopy, Confocal , Microvessels/cytology , Microvessels/drug effects , Particle Size , Polystyrenes/chemistry , Silicon Dioxide/chemistryABSTRACT
BACKGROUND: The chromosomal translocation t(4;11)(q21;q23) is associated with high-risk acute lymphoblastic leukemia of infants. The resulting AF4â¢MLL oncoprotein becomes activated by Taspase1 hydrolysis and is considered to promote oncogenic transcriptional activation. Hence, Taspase1's proteolytic activity is a critical step in AF4â¢MLL pathophysiology. The Taspase1 proenzyme is autoproteolytically processed in its subunits and is assumed to assemble into an αßßα-heterodimer, the active protease. Therefore, we investigated here whether overexpression of catalytically inactive Taspase1 variants are able to interfere with the proteolytic activity of the wild type enzyme in AF4â¢MLL model systems. METHODOLOGY/FINDINGS: The consequences of overexpressing the catalytically dead Taspase1 mutant, Taspase1(T234V), or the highly attenuated variant, Taspase1(D233A), on Taspase1's processing of AF4â¢MLL and of other Taspase1 targets was analyzed in living cancer cells employing an optimized cell-based assay. Notably, even a nine-fold overexpression of the respective Taspase1 mutants neither inhibited Taspase1's cis- nor trans-cleavage activity in vivo. Likewise, enforced expression of the α- or ß-subunits showed no trans-dominant effect against the ectopically or endogenously expressed enzyme. Notably, co-expression of the individual α- and ß-subunits did not result in their assembly into an enzymatically active protease complex. Probing Taspase1 multimerization in living cells by a translocation-based protein interaction assay as well as by biochemical methods indicated that the inactive Taspase1 failed to assemble into stable heterocomplexes with the wild type enzyme. CONCLUSIONS: Collectively, our results demonstrate that inefficient heterodimerization appears to be the mechanism by which inactive Taspase1 variants fail to inhibit wild type Taspase1's activity in trans. Our work favours strategies targeting Taspase1's catalytic activity rather than attempts to block the formation of active Taspase1 dimers to interfere with the pathobiological function of AF4â¢MLL.