Insights in dynamic kinome reprogramming as a consequence of MEK inhibition in MLL-rearranged AML.

Single kinase-targeted cancer therapies often failed prolonged responses because cancer cells bypass through alternative routes. In this study, high-throughput kinomic and proteomic approaches enabled to identify aberrant activity profiles in mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML) that defined druggable targets. This approach revealed impaired activity of proteins belonging to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathway. Pharmacological druggable MAPK pathway targets tested in primary MLL-rearranged AML included MAPKK1/2 (MEK), cyclic AMP-responsive element-binding protein (CREB) and MAPK8/9 (JNK). MEK inhibition showed to severely decrease MLL-rearranged AML cell survival without showing cytotoxicity in normal controls, whereas inhibition of CREB and JNK failed to exhibit MLL selectivity. Exploring the working mechanism of MEK inhibition, we assessed proteome activity in response to MEK inhibition in THP-1. MAPK1/3 (Erk) phosphorylation was instantly decreased in concurrence with a sustained Akt/mammalian target of rapamycin (mTOR) phosphorylation that enabled a subpopulation of cells to survive MEK inhibition. After exhaustion of MEK inhibition the AML cells recovered via increased activity of vascular endothelial growth factor receptor-2 (VEGFR-2) and Erk proteins to resume their proliferative state. Combined MEK and VEGFR-2 inhibition strengthened the reduction in MLL-rearranged AML cell survival by blocking the Akt/mTOR and MAPK pathways simultaneously. The generation of insights in cancerous altered activity profiles and alternative escape mechanisms upon targeted therapy allows the rational design of novel combination strategies.

Vascular endothelial growth factor receptor 2 (VEGFR-2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells.

AIMS: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. METHODS: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. RESULTS: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. CONCLUSIONS: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours.

Effect of aspirin on the Wnt/beta-catenin pathway is mediated via protein phosphatase 2A.

Nonsteroidal anti-inflammatory drugs show chemopreventive efficacy in colon cancer, but the mechanism behind this remains unclear. Elucidating this mechanism is seen as vital to the development of new chemopreventive agents. We studied the effects of aspirin on the oncogenic Wnt/beta-catenin pathway activity in colorectal cancer cell lines and observed that aspirin dose-dependently decreased the activity of this pathway, as judged by TCF-driven luciferase activity, reduced Wnt target gene expression and increased phosphorylation of beta-catenin by immunoblotting. Furthermore, the ubiquitination and cytoplasmic levels of beta-catenin were assessed by immunoblotting, and also the localization of beta-catenin was shown by green fluorescent protein-tagged beta-catenin and time-lapse fluorescent imaging. Importantly, aspirin treatment caused increased phosphorylation of protein phosphatase 2A (PP2A), an event associated with inhibition of PP2A enzymatic activity, which was confirmed by a reduction in enzymatic PP2A activity. Moreover, this inhibition of PP2A enzymatic activity was essential for the effects of aspirin on the Wnt/beta-catenin pathway as shown by transient transfection with PP2A constructs. The findings in this article provide a molecular explanation for the efficacy of aspirin in chemoprevention of colorectal cancer and shows biochemical evidence that PP2A is an important regulator of Wnt/beta-catenin pathway activity in these cells.

Lipopolysaccharide recognition, internalisation, signalling and other cellular effects.

Despite the importance of bacterial lipopolysaccharide (LPS) in infection and inflammation, many aspects of LPS action remain poorly understood. Especially, the mechanisms by which cells recognise and react to endotoxins or endotoxin-containing particles and how cellular responses are translated into systemic effects have long remained obscure. However, the recent identification of Toll-like receptors as essential participants in endotoxin signal transduction has provided the first answers in clarifying cellular LPS responses. In this review, we discuss the consequences of the clarification of the cellular effects of LPS. Furthermore, for LPS to exert its effects, it has to be transported to its target cells and be recognised before signalling may be induced, and we shall review the current state of affairs with regard to these recognition processes. Finally, we shall investigate how current knowledge may explain endotoxin neutralisation and subsequent detoxification, either through LPS internalisation or via LPS immobilisation, or through the actions of LPS-binding molecules.

Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens.

Ceramide (Cer) accumulating during the execution phase of apoptosis is generated from plasma membrane sphingomyelin (SM), which gains access to a sphingomyelinase due to phospholipid scrambling (Tepper, A. D., Ruurs, P., Wiedmer, T., Sims, P., Borst, J., and van Blitterswijk, W. J. (2000) J. Cell. Biol. 150, 155-164). To evaluate the functional significance of this Cer pool, we aimed to convert it to glucosylceramide (GlcCer), by constitutive overexpression of glucosylceramide synthase (GCS). Jurkat cells, retrovirally transduced with GCS cDNA, showed a 10-12-fold increase in GCS activity in vitro and a 7-fold elevated basal GlcCer level in vivo. However, Cer accumulating during apoptosis induced by ligation of the death receptor CD95, treatment with the anti-cancer drug etoposide, or exposure to gamma-radiation was not glycosylated by GCS. Likewise, Cer liberated at the plasma membrane by bacterial SMase was not converted by the enzyme. Thus, GCS, located at the Golgi, is topologically segregated from Cer produced in the plasma membrane. In contrast, de novo synthesized Cer as well as an exogenously supplied cell-permeable Cer analog were efficiently glycosylated, apparently due to different Cer topology and distinct physicochemical behavior of the synthetic Cer species, respectively. Exogenous cell-permeable Cer species, despite their conversion by GCS, effectively induced apoptosis. We also observed that GCS activity is down-regulated in cells undergoing apoptosis. In conclusion, GCS can convert de novo synthesized Cer but not SM-derived Cer, and, therefore, the ability of GCS overexpression to protect cells from possible detrimental effects of Cer accumulation is limited.

Factor VIIa/tissue factor-induced signaling via activation of Src-like kinases, phosphatidylinositol 3-kinase, and Rac.

Tissue factor (TF), apart from activating the extrinsic pathway of the blood coagulation, is a principal regulator of embryonic angiogenesis and oncogenic neoangiogenesis, but also influences inflammation, leukocyte diapedesis and tumor progression. The intracellular domain of TF lacks homology to other classes of receptors and hence the signaling mechanism is poorly understood. Here we demonstrate that factor VIIa (the natural ligand for TF) induces the activation of the Src family members c-Src, Lyn, and Yes, and subsequently phosphatidylinositol 3-kinase (PI3K), followed by stimulation of c-Akt/protein kinase B as well as the small GTPases Rac and Cdc42. In turn Rac mediates p38 mitogen-activated protein (MAP) kinase activation and cytoskeletal reorganization, whereas factor VIIa-induced p42/p44 MAP kinase stimulation required PI3K enzymatic activity but was not inhibited by dominant negative Rac proteins. We propose that this Src family member/PI3K/Rac-dependent signaling pathway is a major mediator of factor VIIa/TF effects in pathophysiology.