ABSTRACT
Recently the Aurora-Kinases (Aurk) moved into the focus as novel disease related biomarkers and therapeutic targets. Elevated Aurora-Kinase expression has been found in a number of malignancies, amongst them HNSCC. For esophageal cancer, the AurkA Phe31-Ile polymorphism has previously been associated with tumor progression. Here we evaluated the treatment efficiency of HNSCC cell radiation as a function of Aurora-Kinases in HNSCC cell lines. Moreover, we investigated a potential sensitization to radiation by a cell treatment with the inhibitors Alisertib, Barasertib, Docetaxel and VX-680. In parallel the radiation dependent expression and regulation of AurkA/B, p-Akt Ser 473 and Survivin and the AurkA polymorphism were investigated in primary tumor samples. We identified a high-risk collective with elevated AurkA and Survivin or AurkA and p-Akt Ser 473 expression. High AurkA, AurkB, and p-Akt Ser 473 expression was exclusively found in the heterozygous cell line. We found a polymorphism dependent sensitivity to treatments with different Aurk inhibitors: The homozygous cell line UD-SCC-5 could be sensitized to radiation with Docetaxel in combination with any of the Aurora-Kinase inhibitors. In contrast, treatment with Docetaxel or radiation did not enhance the inhibitory effect of Barasertib or VX-680 in the heterozygous SAS cell line. These findings indicate that the Aurora-Kinase A Phe31-Ile-polymorphism is a possibly predictive factor for response to radiation in combination with Docetaxel and Aurora-Kinase inhibitor treatments.
ABSTRACT
BACKGROUND: The epidermal growth factor receptor (EGFR) is an important regulator of cell growth and survival, and is highly variable in tumor cells. The most prevalent variation of the EGFR extracellular domain is the EGFR variant III (EGFRvIII). Some studies imply that EGFRvIII may be responsible for the poor response to the monoclonal EGFR-antibody Cetuximab, used therapeutically in head and neck squamous cell carcinoma (HNSCC). Due to inconsistent data in the literature regarding EGFRvIII prevalence and clinical relevance in HNSCC, especially its predictive value, we examined EGFRvIII-transfected cell lines and patient tissue samples. RESULTS: In contrast to other recent publications, we were able to demonstrate EGFRvIII expression in HNSCC. However, we noted that the different detection methods yielded inconsistent results. Furthermore, our EGFRvIII transfected and EGFR wild type cell lines exhibited similar characteristics and response rates in the performed in vitro experiments. MATERIALS AND METHODS: We conducted various inhibition and combined irradiation experiments using three EGFRvIII-transfected cell lines. Moreover, a patient cohort of 149 cases consisting of formalin fixed and paraffin embedded (FFPE) and fresh-frozen specimens was assayed via reverse transcriptase PCR (rtPCR) with gel electrophoresis and sequencing for EGFRvIII prevalence. In the rtPCR assays, we used five previously published EGFRvIII primers and EGFRvIII-positive glioblastoma tissue as a positive control. In addition, immunohistochemical staining was conducted. CONCLUSIONS: EGFRvIII can be detected in HNSCC patient samples. Nevertheless, the low prevalence and similar response rates to targeted drugs and irradiation in vitro cast doubt regarding the clinical relevance of EGFRvIII in HNSCC.
Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , Carcinoma, Squamous Cell/therapy , Cetuximab/therapeutic use , ErbB Receptors/genetics , Head and Neck Neoplasms/therapy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/radiation effects , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Cohort Studies , ErbB Receptors/metabolism , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , Molecular Targeted Therapy , Paraffin Embedding , Radiotherapy , Squamous Cell Carcinoma of Head and Neck , Tissue Fixation , TransfectionABSTRACT
In higher eukaryotes, PAPS synthases are the only enzymes producing the essential sulphate-donor 3'-phospho-adenosine-5'-phosphosulphate (PAPS). Recently, PAPS synthases have been associated with several genetic diseases and retroviral infection. To improve our understanding of their pathobiological functions, we analysed the intracellular localisation of the two human PAPS synthases, PAPSS1 and PAPSS2. For both enzymes, we observed pronounced heterogeneity in their subcellular localisation. PAPSS1 was predominantly nuclear, whereas PAPSS2 localised mainly within the cytoplasm. Treatment with the nuclear export inhibitor leptomycin B had little effect on their localisation. However, a mutagenesis screen revealed an Arg-Arg motif at the kinase interface exhibiting export activity. Notably, both isoforms contain a conserved N-terminal basic Lys-Lys-Xaa-Lys motif indispensable for their nuclear localisation. This nuclear localisation signal was more efficient in PAPSS1 than in PAPSS2. The activities of the identified localisation signals were confirmed by microinjection studies. Collectively, we describe unusual localisation signals of both PAPS synthase isoforms, mobile enzymes capable of executing their function in the cytoplasm as well as in the nucleus.
Subject(s)
Cell Nucleus/enzymology , Multienzyme Complexes/metabolism , Sulfate Adenylyltransferase/metabolism , Active Transport, Cell Nucleus/drug effects , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Nucleus/drug effects , Conserved Sequence/genetics , Cytosol/enzymology , Fatty Acids, Unsaturated/pharmacology , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Karyopherins/metabolism , Mice , Molecular Sequence Data , Molecular Weight , Multienzyme Complexes/chemistry , Mutagenesis/drug effects , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Nuclear Export Signals/drug effects , Nuclear Localization Signals/chemistry , Protein Transport/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/drug effects , Subcellular Fractions/enzymology , Sulfate Adenylyltransferase/chemistry , Exportin 1 ProteinABSTRACT
We recently showed that the activity of the 2-oxoglutarate dehydrogenase complex (ODHC) in Corynebacterium glutamicum is controlled by a novel regulatory mechanism that involves a 15-kDa protein called OdhI and serine/threonine protein kinase G (PknG). In its unphosphorylated state, OdhI binds to the E1 subunit (OdhA) of ODHC and, thereby, inhibits its activity. Inhibition is relieved by phosphorylation of OdhI at threonine-14 by PknG under conditions requiring high ODHC activity. In this work, evidence is provided that the dephosphorylation of phosphorylated OdhI is catalyzed by a phospho-Ser/Thr protein phosphatase encoded by the gene cg0062, designated ppp. As a decreased ODHC activity is important for glutamate synthesis, we investigated the role of OdhI and PknG for glutamate production under biotin limitation and after addition of Tween-40, penicillin, or ethambutol. A DeltaodhI mutant formed only 1-13% of the glutamate synthesized by the wild type. Thus, OdhI is essential for efficient glutamate production. The effect of a pknG deletion on glutamate synthesis was dependent on the induction conditions. Under strong biotin limitation and in the presence of ethambutol, the DeltapknG mutant showed significantly increased glutamate production, offering a new way to improve production strains.
