[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Cellular and Molecular Medicine 01/2013; · 4.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF). By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.
PLoS ONE 01/2013; 8(9):e73473. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The type-II-cytokine IFN-gamma is a pivotal player in innate immune responses but also assumes functions in controlling tumor cell growth by orchestrating cellular responses against neoplastic cells. The role of IFN-gamma in melanoma is not fully understood: it is a well-known growth inhibitor of melanoma cells in vitro. On the other hand, IFN-gamma may also facilitate melanoma progression. While interferon-regulated genes encoding proteins have been intensively studied since decades, the contribution of miRNAs to effects mediated by interferons is an emerging area of research.We recently described a distinct and dynamic regulation of a whole panel of microRNAs (miRNAs) after IFN-gamma-stimulation. The aim of this study was to analyze the transcriptional regulation of miR-29 family members in detail, identify potential interesting target genes and thus further elucidate a potential signaling pathway IFN-gamma [rightwards arrow] Jak[rightwards arrow] P-STAT1 [rightwards arrow] miR-29 [rightwards arrow] miR-29 target genes and its implication for melanoma growth. RESULTS: Here we show that IFN-gamma induces STAT1-dependently a profound up-regulation of the miR-29 primary cluster pri-29a~b-1 in melanoma cell lines. Furthermore, expression levels of pri-29a~b-1 and mature miR-29a and miR-29b were elevated while the pri-29b-2~c cluster was almost undetectable. We observed an inverse correlation between miR-29a/b expression and the proliferation rate of various melanoma cell lines. This finding could be corroborated in cells transfected with either miR-29 mimics or inhibitors. The IFN-gamma-induced G1-arrest of melanoma cells involves down-regulation of CDK6, which we proved to be a direct target of miR-29 in these cells. Compared to nevi and normal skin, and metastatic melanoma samples, miR-29a and miR-29b levels were found strikingly elevated in certain patient samples derived from primary melanoma. CONCLUSIONS: Our findings reveal that the miR-29a/b1 cluster is to be included in the group of IFN- and STAT-regulated genes. The up-regulated miR-29 family members may act as effectors of cytokine signalling in melanoma and other cancer cells as well as in the immune system.
Cell Communication and Signaling 12/2012; 10(1):41. · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are ubiquitously expressed small, non-coding RNAs that negatively regulate gene expression at a post-transcriptional level. So far, over 1000 miRNAs have been identified in human cells and their diverse functions in normal cell homeostasis and many different diseases have been thoroughly investigated during the past decade. MiR-29, one of the most interesting miRNA families in humans to date, consists of three mature members miR-29a, miR-29b and miR-29c, which are encoded in two genetic clusters. Members of this family have been shown to be silenced or down-regulated in many different types of cancer and have subsequently been attributed predominantly tumor-suppressing properties, albeit exceptions have been described where miR-29s have tumor-promoting functions. MiR-29 targets expression of diverse proteins like collagens, transcription factors, methyltransferases and others, which may partake in abnormal migration, invasion or proliferation of cells and may favor development of cancer. Furthermore, members of the miR-29 family can be activated by interferon signaling, which suggests a role in the immune system and in host-pathogen interactions, especially in response to viral infections. In this review, we summarize current knowledge on the genomic organization and regulation of the miR-29 family and we provide an overview of its implication in cancer suppression and promotion as well as in host immune responses. The numerous remarkable properties of these miRNAs and their often altered expression patterns might make the miR-29 family promising biomarkers and therapeutic targets for various diseases in future.
Current Molecular Medicine 08/2012; · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs are major players in post-transcriptional gene regulation. Even small changes in miRNA levels may have profound consequences for the expression levels of target genes. Hence, miRNAs themselves need to be tightly, albeit dynamically, regulated. Here, we investigated the dynamic behavior of miRNAs over a wide time range following stimulation of melanoma cells with interferon-γ (IFN-γ), which activates the transcription factor STAT1. By applying several bioinformatic and statistical software tools for visualization and identification of differentially expressed miRNAs derived from time-series microarray experiments, 8.9% of 1105 miRNAs appeared to be directly or indirectly regulated by STAT1. Focusing on distinct dynamic expression patterns, we found that the majority of robust miRNA expression changes occurred in the intermediate time range (24-48 h). Three miRNAs (miR-27a, miR-30a, miR-34a) had a delayed regulation occurring at 72 h while none showed significant expression changes at early time points between 30 min and 6 h. Expression patterns of individual miRNAs were altered gradually over time or abruptly increased or decreased between two time points. Furthermore, we observed coordinated dynamic transcription of most miRNA clusters while few were found to be regulated independently of their genetic cluster. Most interestingly, several "star" or passenger strand sequences were specifically regulated over time while their "guide" strands were not.
[Show abstract][Hide abstract] ABSTRACT: Genetic deficiency of Jak3 leads to abrogation of signal transduction through the common gamma chain (γc) and thus to immunodeficiency suggesting that specific inhibition of Jak3 kinase may result in immunosuppression. Jak1 cooperates with Jak3 in signaling through γc-containing receptors. Unexpectedly, a Jak3-selective inhibitor was less efficient in abolishing STAT5 phosphorylation than pan-Jak inhibitors. We therefore explored the roles of Jak1 and Jak3 kinase functionality in signaling using a reconstituted system. The presence of kinase-inactive Jak1 but not kinase-inactive Jak3 resulted in complete abolishment of STAT5 phosphorylation. Specific inhibition of the "analog-sensitive" mutant AS-Jak1 but not AS-Jak3 by the ATP-competitive analog 1NM-PP1 abrogated IL-2 signaling, corroborating the data with the selective Jak3 inhibitor. Jak1 thus plays a dominant role over Jak3 and these data challenge the notion that selective ATP-competitive Jak3 kinase inhibitors will be effective.
[Show abstract][Hide abstract] ABSTRACT: OSM, a cytokine of the IL-6-type cytokine family, regulates inflammatory processes (like the acute phase response), tissue remodeling, angiogenesis, cell differentiation and proliferation. Inflammation is discussed to favor carcinogenesis and the inflammatory cytokine OSM was lately described to up-regulate HIF-1α, whose up-regulation is also observed in many cancers. In this study we demonstrate that OSM, and to a lesser degree IL-6, induces the expression of Grp78/BiP, an ER chaperone associated with tumor development and poor prognosis in cancer. In contrast, IFN-γ or TNF-α had no effect on Grp78 expression. The up-regulation seems to be specific to liver cells, as it occurs in hepatocytes and hepatoma cells but not in prostate, melanoma, breast or kidney cells. OSM does not lead to up-regulation of Grp94, enhanced XBP-1 mRNA splicing or phosphorylation of eIF2α, indicating that it is not associated to a general ER stress response. Analysis of the underlying mechanism showed that Grp78 is up-regulated by transcriptional processes which are to the greater part, though not completely, dependent on MEK/Erk activation.
[Show abstract][Hide abstract] ABSTRACT: Small noncoding microRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation or orchestrating their sequence-specific degradation. In this study, we investigated miRNA and miRNA target gene expression patterns in melanoma to identify candidate biomarkers for early and progressive disease. Because data presently available on miRNA expression in melanoma are inconsistent thus far, we applied several different miRNA detection and profiling techniques on a panel of 10 cell lines and 20 patient samples representing nevi and primary or metastatic melanoma. Expression of selected miRNAs was inconsistent when comparing cell line-derived and patient-derived data. Moreover, as expected, some discrepancies were also detected when miRNA microarray data were correlated with qPCR-measured expression levels. Nevertheless, we identified miRNA-200c to be consistently downregulated in melanocytes, melanoma cell lines, and patient samples, whereas miRNA-205 and miRNA-23b were markedly reduced only in patient samples. In contrast, miR-146a and miR-155 were upregulated in all analyzed patients but none of the cell lines. Whole-genome microarrays were performed for analysis of selected melanoma cell lines to identify potential transcriptionally regulated miRNA target genes. Using Ingenuity pathway analysis, we identified a deregulated gene network centered around microphthalmia-associated transcription factor, a transcription factor known to play a key role in melanoma development. Our findings define miRNAs and miRNA target genes that offer candidate biomarkers in human melanoma.
Cancer Research 05/2010; 70(10):4163-73. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets.
Journal of Cellular and Molecular Medicine 03/2010; 14(3):504-27. · 4.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1beta, TNFalpha or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.
[Show abstract][Hide abstract] ABSTRACT: The 13th meeting of the Signal Transduction Society was held in Weimar, from October 28 to 30, 2009. Special focus of the 2009 conference was "Aging and Senescence", which was co-organized by the SFB 728 "Environmentally-Induced Aging Processes" of the University of Düsseldorf and the study group 'Signal Transduction' of the German Society for Cell Biology (DGZ). In addition, several other areas of signal transduction research were covered and supported by different consortia associated with the Signal Transduction Society including the long-term associated study groups of the German Society for Immunology and the Society for Biochemistry and Molecular Biology, and for instance the SFB/Transregio 52 "Transcriptional Programming of Individual T Cell Subsets" located in Würzburg, Mainz and Berlin. The different research areas that were introduced by outstanding keynote speakers attracted more than 250 scientists, showing the timeliness and relevance of the interdisciplinary concept and exchange of knowledge during the three days of the scientific program. This report gives an overview of the presentations of the conference.
Cell Communication and Signaling 02/2010; 8:2. · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia.
[Show abstract][Hide abstract] ABSTRACT: The interleukin-6-type cytokine oncostatin M (OSM) acts via the Janus kinase/signal transducer and activator of transcription pathway as well as via activation of mitogen-activated protein kinases and is known to critically regulate processes such as liver development and regeneration, hematopoiesis, and angiogenesis, which are also determined by hypoxia with the hypoxia-inducible factor 1alpha (HIF1alpha) as a key component. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF1alpha under normoxic and hypoxic conditions. Furthermore, the OSM-dependent HIF1alpha increase is mediated via Janus kinase/signal transducer and activator of transcription 3 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 pathways. OSM-mediated HIF1alpha up-regulation did not result from an increase in HIF1alpha protein stability but from increased transcription from the HIF1alpha gene. In addition, we show that the OSM-induced HIF1alpha gene transcription and the resulting enhanced HIF1alpha protein levels are important for the OSM-dependent vascular endothelial growth factor and plasminogen activator inhibitor 1 gene induction associated with several diseases. Conclusion: HIF1alpha levels increase significantly after treatment of hepatocytes and hepatoma cells with OSM, and HIF1alpha contributes to OSM downstream signaling events, pointing to a cross-talk between cytokine and hypoxia signaling in processes such as liver development and regeneration.
[Show abstract][Hide abstract] ABSTRACT: The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated in myeloproliferative diseases and leukemia. Although Jaks were studied extensively for many years, the molecular mechanism of Jak activation upon cytokine stimulation of cells is still incompletely understood. In this study, we investigated the importance of an unusual insertion located within the kinase domain in Jak2. We found that the deletion of this insertion, which we named the Jak-specific insertion (JSI), totally abrogates Jak2 autophosphorylation. We further point mutated four residues within the JSI that are conserved in all Jak family members. Three of these mutants showed abrogated or reduced autophosphorylation, whereas the fourth displayed increased autophosphorylation. We found that the phosphorylation state of these mutants is not influenced by other domains of the kinase. Our data further suggest that the JSI is not required for the negative regulation of kinase activity by the suppressor of cytokine signaling proteins, SOCS. Most importantly, we show that mutations in this region differentially affect IFN-gamma and erythropoietin signal transduction. Taken together, the dramatic effects on the phosphorylation status of Jak2 as well as the differential effects on the signaling via different cytokines highlight the importance of this unusual region for the catalytic activity of Jaks.
The Journal of Immunology 04/2009; 182(5):2969-77. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IL-24, a member of the IL-10 family of cytokines, is produced by monocytes and Th2 cells. Interestingly, immune cells do not appear to express specific IL-24 receptor chains (IL-20R1/IL-20R2 and IL-22R/IL-20R2), it is therefore unlikely that IL-24 has classical immune-modulating properties. Skin, on the other hand, seems to represent a major target tissue for IL-24 and related cytokines such as IL-19, -20, and -22. However, the initial interest in IL-24 did not arise from its physiological signalling properties through its cognate receptors but rather because of its tentative ability to selectively kill different cancer cells. In an attempt to further investigate the signalling events underlying the IL-24-induced cancer cell death, we found that melanoma cell lines did not react in the expected and previously described way. Using several different forms and delivery modes of IL-24, we were unable to detect any apoptosis-inducing properties of this cytokine in melanoma cells. In the present 'Point of view' we will briefly summarize these findings and put them in context of published reports stating that IL-24 might be a long sought after treatment for several types of cancer.
Journal of Cellular and Molecular Medicine 12/2008; 12(6A):2505-10. · 4.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1, ezrin, radixin, moesin (FERM) domain at their N terminus. FERM domains are composed of three structurally unrelated subdomains (F1, F2, and F3) which are in close contact to one another and form the clover-shaped FERM domain. We generated a model structure of the Jak1 FERM domain, based on solved FERM structures and the alignments with other FERM domains. To destabilize different subdomains and to uncover their exact function, we mutated specific hydrophobic residues conserved in FERM domains and involved in hydrophobic core interactions. In this study, we show that the structural integrity of the F2 subdomain of the FERM domain of Jak1 is necessary to bind the IFN-gammaRalpha. By mutagenesis of hydrophobic residues in the hydrophobic core between the three FERM subdomains, we find that the structural context of the FERM domain is necessary for the inhibition of Jak1 phosphorylation. Thus, FERM domain mutations can have repercussions on Jak1 function. Interestingly, a mutation in the kinase domain (Jak1-K907E), known to abolish the catalytic activity, also leads to an impaired binding to the IFN-gammaRalpha when this mutant is expressed at endogenous levels in U4C cells. Our data show that the structural integrity of both the FERM domain and of the kinase domain is essential for both receptor binding and catalytic function/autoinhibition.
The Journal of Immunology 02/2008; 180(2):998-1007. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Signal transducers and activators of transcriptions (STAT) are key mediators of cytokine signaling. Moreover, these transcription factors play a crucial role in oncogenic signaling where inappropriate and sustained activation of STATs, especially STAT3, is a trait of many different cancers and their derived cell lines. Constitutively active STAT3 has been reported to prevent programmed cell death and enhance cell proliferation, whereas the disruption of STAT3 signaling can inhibit tumor growth. The physiologic activation of STAT3 by cytokines has been well established; however, little is known about altered, stimulation-independent STAT3 activation. Here, we show that, in most but not all melanoma cell lines, STAT3 phosphorylation increased substantially with cell density and that this STAT3 was able to bind to DNA and to activate transcription. Inhibitor studies showed that the cell density-dependent STAT3 activation relies on Janus kinases (JAK) rather than Src kinases. Using a specific JAK inhibitor, sustained STAT3 activation was completely abrogated in all tested melanoma lines, whereas inhibition of Src or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 had no effect on constitutively tyrosine-phosphorylated STAT3 levels. Although STAT3 activation was completely blocked with JAK inhibitor I and to a lesser extent with the common JAK inhibitor AG490, only the latter compound markedly decreased proliferation and induced apoptosis. Taken together, variations in cell density can profoundly modify the extent of JAK-mediated persistent STAT3 phosphorylation; however, STAT3 activation was not sufficient to provide critical growth and survival signals in melanoma cell lines.
Molecular Cancer Research 01/2008; 5(12):1331-41. · 4.35 Impact Factor