Hypoxia-responsive miR-210 promotes self-renewal capacity of colon tumor-initiating cells by repressing ISCU and by inducing lactate production.

Low oxygen concentrations (hypoxia) are known to affect the cellular metabolism and have been suggested to regulate a subpopulation of cancer cells with tumorigenic properties, the so-called tumor-initiating cells (TICs). To better understand the mechanism of hypoxia-induced TIC activation, we set out to study the role of hypoxia-responsive miRNAs in recently established colon cancer patient-derived TICs. We were able to show that low oxygen concentrations consistently lead to the upregulation of miR-210 in different primary TIC-enriched cultures. Both stable overexpression of miR-210 and knockdown of its target gene ISCU resulted in enhanced TIC self-renewal. We could validate the tumorigenic properties of miR- 210 in in vivo experiments by showing that ectopic expression of miR-210 results in increased tumor incidence. Furthermore, enhanced miR-210 expression correlated with reduced TCA cycle activity and increased lactate levels. Importantly, by blocking lactate production via inhibition of LDHA, we could reverse the promoting effect of miR-210 on self-renewal capacity, thereby emphasizing the regulatory impact of the glycolytic phenotype on colon TIC properties. Finally, by assessing expression levels in patient tissue, we could demonstrate the clinical relevance of the miR-210/ISCU signaling axis for colorectal carcinoma. Taken together, our study highlights the importance of hypoxia-induced miR-210 in the regulation of colon cancer initiation.

L-plastin Ser5 phosphorylation in breast cancer cells and in vitro is mediated by RSK downstream of the ERK/MAPK pathway.

Deregulated cell migration and invasion are hallmarks of metastatic cancer cells. Phosphorylation on residue Ser5 of the actin-bundling protein L-plastin activates L-plastin and has been reported to be crucial for invasion and metastasis. Here, we investigate signal transduction leading to L-plastin Ser5 phosphorylation using 4 human breast cancer cell lines. Whole-genome microarray analysis comparing cell lines with different invasive capacities and corresponding variations in L-plastin Ser5 phosphorylation level revealed that genes of the ERK/MAPK pathway are differentially expressed. It is noteworthy that in vitro kinase assays showed that ERK/MAPK pathway downstream ribosomal protein S6 kinases α-1 (RSK1) and α-3 (RSK2) are able to directly phosphorylate L-plastin on Ser5. Small interfering RNA- or short hairpin RNA-mediated knockdown and activation/inhibition studies followed by immunoblot analysis and computational modeling confirmed that ribosomal S6 kinase (RSK) is an essential activator of L-plastin. Migration and invasion assays showed that RSK knockdown led to a decrease of up to 30% of migration and invasion of MDA-MB-435S cells. Although the presence of L-plastin was not necessary for migration/invasion of these cells, immunofluorescence assays illustrated RSK-dependent recruitment of Ser5-phosphorylated L-plastin to migratory structures. Altogether, we provide evidence that the ERK/MAPK pathway is involved in L-plastin Ser5 phosphorylation in breast cancer cells with RSK1 and RSK2 kinases able to directly phosphorylate L-plastin residue Ser5.

JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells.

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner.

JAK2 mutants (e.g., JAK2V617F) and their importance as drug targets in myeloproliferative neoplasms.

The Janus kinase 2 (JAK2) mutant V617F and other JAK mutants are found in patients with myeloproliferative neoplasms and leukemias. Due to their involvement in neoplasia and inflammatory disorders, Janus kinases are promising targets for kinase inhibitor therapy. Several small-molecule compounds are evaluated in clinical trials for myelofibrosis, and ruxolitinib (INCB018424, Jakafi®) was the first Janus kinase inhibitor to receive clinical approval. In this review we provide an overview of JAK2V617F signaling and its inhibition by small-molecule kinase inhibitors. In addition, myeloproliferative neoplasms are discussed regarding the role of JAK2V617F and other mutant proteins of possible relevance. We further give an overview about treatment options with special emphasis on possible combination therapies.

Cooperative effects of Janus and Aurora kinase inhibition by CEP701 in cells expressing Jak2V617F.

The Janus kinase 2 mutant V617F occurs with high frequency in myeloproliferative neoplasms. Further mutations affecting the Janus kinase family have been discovered mostly in leukaemias and in myeloproliferative neoplasms. Owing to their involvement in neoplasia, inflammatory diseases and in the immune response, Janus kinases are promising targets for kinase inhibitor therapy in these disease settings. Various quantitative assays including two newly developed screening assays were used to characterize the function of different small-molecule compounds in cells expressing Jak2V617F. A detailed comparative analysis of different Janus kinase inhibitors in our quantitative assays and the subsequent characterization of additional activities demonstrated for the first time that the most potent Jak2 inhibitor in our study, CEP701, also targets Aurora kinases. CEP701 shows a unique combination of both activities which is not found in other compounds also targeting Jak2. Furthermore, colony forming cell assays showed that Janus kinase 2 inhibitors preferentially suppressed the growth of erythroid colonies, whereas inhibitors of Aurora kinases preferentially blocked myeloid colony growth. CEP701 demonstrated a combined suppression of both colony types. Moreover, we show that combined application of a Janus and an Aurora kinase inhibitor recapitulated the effect observed for CEP701 but might allow for more flexibility in combining both activities in clinical settings, e.g. in the treatment of myeloproliferative neoplasms. The newly developed screening assays are high throughput compatible and allow an easy detection of new compounds with Janus kinase 2 inhibitory activity.

Effects of Staphylococcus aureus-hemolysin A on calcium signalling in immortalized human airway epithelial cells.

Part of the innate defence of bronchial epithelia against bacterial colonization is secretion of salt and water which generally depends on coordinated actions of receptor-mediated cAMP- and calcium signalling. The hypothesis that Staphylococcus aureus-virulence factors interfere with endogenous signals in host cells was tested by measuring agonist-mediated changes in [Ca(2+)](i) in S9 cells upon pre-incubation with bacterial secretory products. S9 cells responded to mAChR-activation with calcium release from intracellular stores and capacitative calcium influx. Treatment of cells with culture supernatants of S. aureus (COL) or with recombinant alpha-hemolysin (Hla) resulted in time- and concentration-dependent changes in [Ca(2+)](i). High concentrations of Hla (2000 ng/ml) resulted in elevations in [Ca(2+)](i) elicited by accelerated calcium influx. A general Hla-mediated permeabilization of S9 cell membranes to small molecules, however, did not occur. Lower concentrations of Hla (200 ng/ml) induced a reduction in [Ca(2+)](i)-levels during the sustained plateau phase of receptor-mediated calcium signalling which was abolished by pre-incubation of cells with carboxyeosin, an inhibitor of the plasma membrane calcium-ATPase. This indicates that low concentrations of Hla change calcium signalling by accelerating pump-driven extrusion of Ca(2+) ions. In vivo, such a mechanism may result in attenuation of calcium-mediated cellular defence functions and facilitation of bacterial adherence to the bronchial epithelium.

Phospholipase C-activating plasma membrane receptors and calcium signaling in immortalized human airway epithelial cells.

Mechanical clearance of inhaled dust particles and microorganisms is an important part of the innate defense mechanisms of mammalian airways. Airway epithelia are composed of various cell types with different degrees of cell polarity. Serous cells regulate composition and volume of luminal periciliary fluid and mucus. Autocrine, paracrine, or neuronal messengers determine the secretory and reabsorptive rates of electrolytes and water via cAMP-or inositol triphosphate/calcium-mediated intracellular signals. Comparison of the expression of calcium-mobilizing receptor types (G protein-coupled-, growth factor-, and cytokine receptors) in two types of human immortalized airway epithelial cells (S9, 16HBE14o-) revealed that receptor populations were qualitatively and quantitatively different in the two cell types. Sustained calcium signals were elicited by activation of purinergic receptors in 16HBE14o-cells or muscarinic acetylcholine or histamine receptors in S9 cells. These G protein-coupled receptors mobilized calcium from intracellular stores and activated capacitative calcium influx. The experimental cells may represent different types of original airway epithelial cells and seem to be suited as model cells to study cell signaling and protein expression during interaction with pathogens or their secretory products (e.g., virulence factors).