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Aberrant activation of oncogenic kinases is frequently observed in human cancers, but the underlying mechanism and resulting effects on global signaling are incompletely understood. Here, we demonstrate that the oncogenic FIP1L1-PDGFRα kinase exhibits a significantly different signaling pattern compared to its PDGFRα wild type counterpart. Interestingly, the activation of primarily membrane-based signal transduction processes (such as PI3-kinase- and MAP-kinase- pathways) is remarkably shifted toward a prominent activation of STAT factors. This diverging signaling pattern compared to classical PDGF-receptor signaling is partially coupled to the aberrant cytoplasmic localization of the oncogene, since membrane targeting of FIP1L1-PDGFRα restores activation of MAPK- and PI3K-pathways. In stark contrast to the classical cytokine-induced STAT activation process, STAT activation by FIP1L1-PDGFRα does neither require Janus kinase activity nor Src kinase activity. Furthermore, we investigated the mechanism of STAT5 activation via FIP1L1-PDGFRα in more detail and found that STAT5 activation does not involve an SH2-domain-mediated binding mechanism. We thus demonstrate that STAT5 activation occurs via a non-canonical activation mechanism in which STAT5 may be subject to a direct phosphorylation by FIP1L1-PDGFRα. [ABSTRACT FROM PUBLISHER]

The presence of a Src homology 2 (SH2) domain sequence similarity in the sequence of Janus kinases (Jaks) has been discussed since the first descriptions of these enzymes. We performed an in depth study to determine the function of the Jak1 SH2 domain. We investigated the functionality of the Jak1 SH2 domain by stably reconstituting Jak1-defective human fibrosarcoma cells U4C with endogenous amounts of Jak1 in which the crucial arginine residue Arg466 within the SH2 domain has been replaced by lysine. This mutant still binds to the receptor subunits gp130 and OSMR. Moreover, the SH2 R466K mutation does not affect the subcellular distribution of Jak1 as assessed by cell fractionation and confocal microscopy of cells expressing endogenous levels of non-tagged or a yellow fluorescent protein (YFP).tagged Jak1-R466K, respectively. Likewise, the signaling capacity of Jak1 was not affected by this point mutation. However, we found that the SH2 domain is structurally important for cytokine receptor binding and surface expression of the OSMR. [ABSTRACT FROM AUTHOR]

IL-6 plays important roles in regulation of liver functions and promotes hepatocarcinogenesis. As the contribution of IL-6-induced miRNAs to these effects is largely unknown, we investigate the effects of IL-6 on the miRNome of hepatoma cells and non-transformed hepatocytes. Moreover, little is known about miRNAs regulating key players of IL-6 signal transduction. Therefore, our aims were to identify such miRNAs in a miRNA mimics screen and to elucidate their effects on this pathway. Integration of the results of both approaches is expected to lead to the identification of regulatory circuits. While IL-6 (and hyperIL-6) induce the differential regulation of thousands of mRNAs in HepG2 and HuH-7 hepatoma cell lines, only selected miRNAs change their expression level significantly, including miR-21, miR-100, miR-145, miR-146b-5p. In contrast, we have observed previously that IFN-g has a profound effect on the miRNome of melanoma cells (Schmitt, Cell Comm. Sign. 2012). Therefore we investigate whether IL-6-type cytokines may have generally weaker effects on the miRNome compared to interferons and/or whether our observation is due to cell type-specific differences. To extend our in vitro findings, we analyse and correlate the expression levels of miRNAs and inflammatory cytokines in patients with HCC and advanced nonalcoholic steatohepatitis (NASH), the latter being at high risk to develop HCC. Bio-Plex cytokine immunoassays revealed that the serum level of IL-6 and HGF is higher in HCC patients than in healthy controls and NASH patients. Our work may contribute to the identification of novel biomarkers for the progression of chronic liver diseases. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC), mostly the result of chronic liver diseases, is ranked as the fifth most common cancer world-wide. To find new treatments, a better understanding of the initiation, the progression and the implication of the cellular metabolism in the development of HCC is needed. Hypoxia and IL6-type cytokines can promote tumour growth, influence cellular metabolism and prevent cancer cells from apoptosis [1] . Furthermore, IL6-type cytokines induce the expression of HIF-1 α , one of the key mediators of the cellular response to hypoxia [2] . Constitutively active STAT3 leads to an increased production of lactate under normoxic conditions. This effect was HIF-1 α -dependent [3] . We investigated the effects of the IL6-type cytokine Oncostatin M (OSM) on hepatocytes and HCC cell lines under normoxia. Quantitative real-time PCR and quantitative WB analysis was performed focusing on the regulation of glycolytic enzymes, many being direct HIF-1 α target genes. Contrary to the HCC lines, in non-neoplastic PH5CH8 hepatocytes an OSM-mediated induction of pyruvate dehydrogenase kinase 1 (PDK1) was found together with an increase in the pyruvate dehydrogenase (PDH) phosphorylation level, indicating that these cells shift to a more glycolytic metabolism. Metabolic analysis is being carried out to validate this finding. We found little OSM effects under normoxia on the induction of glycolytic enzymes (mRNA and protein level). Surprisingly, even hypoxic treatment only affected few genes previously described to be HIF-1 α targets. Funded by the University of Luxembourg Tandem project “Meta-IL6”. [ABSTRACT FROM AUTHOR]

Interleukin-27 (IL-27) is a type-I-cytokine predominantly secreted by activated macrophages and dendritic cells. Best described are the effects of IL-27 on T-cells and on innate immune cells. Although IL-27 can have pro-inflammatory effects, many studies also suggest that IL-27 is immunosuppressive. IL-27 is composed of the two non-covalently linked subunits p28 and EBI3 (Epstein-Barr virus induced gene 3) and signals via the WSX-1 and gp130 cytokine receptors; it can be grouped into the IL-6/IL-12 superfamily of cytokines. We here report that a variety of non-hematopoietic cells respond to IL-27. When comparing signaling of IL-27 to the one of IL-6-type cytokines (STAT3-dominated) and Interferon-gamma (STAT1-dominated), we observe that IL-27 elicits responses similar to Interferon-gamma. [ABSTRACT FROM AUTHOR]

The activating JAK2-V617F mutation is found in many patients suffering from Myeloproliferative neoplasms such as Polycythemia Vera and Essential Thrombocythemia. JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. We demonstrate uncontrolled membrane binding of the multi-site docking protein Gab1 in JAK2-V617F expressing cells. We elaborated the molecular mechanism and regulation of Gab1-membrane recruitment. In line with the regulatory function of Gab1 on STAT-independent signalling we show that PI3K signalling regulates MAPK activation in JAK2-V617F-positive 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. In summary, our study demands to consider cross-talk mechanisms as potential new targets. This may increase the specificity for intervention approaches compared to the interference in very upstream signalling elements such as receptor kinases or receptor-associated kinases. [ABSTRACT FROM AUTHOR]

Melanomas frequently metastasize to the brain. Despite recent progress in the treatment of melanoma brain metastasis, therapy resistance and relapse of disease remain unsolved challenges. CCT196969 is a SRC family kinase (SFK) and Raf proto-oncogene, serine/threonine kinase (RAF) inhibitor with documented effects in primary melanoma cell lines in vitro and in vivo. Using in vitro cell line assays, we studied the effects of CCT196969 in multiple melanoma brain metastasis cell lines. The drug effectively inhibited proliferation, migration, and survival in all examined cell lines, with viability IC50 doses in the range of 0.18-2.6 μM. Western blot analysis showed decreased expression of p-ERK, p-MEK, p-STAT3 and STAT3 upon CCT196969 treatment. Furthermore, CCT196969 inhibited viability in two B-Raf Proto-Oncogene (BRAF) inhibitor resistant metastatic melanoma cell lines. Further in vivo studies should be performed to determine the treatment potential of CCT196969 in patients with treatment-naïve and resistant melanoma brain metastasis., Competing Interests: The authors declare that no competing interests exist.

Interleukin-6 (IL-6)-type cytokines share the common receptor glycoprotein 130 (gp130), which activates a signaling cascade involving Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) transcription factors. IL-6 and/or its signaling pathway is often deregulated in diseases, such as chronic liver diseases and cancer. Thus, the identification of compounds inhibiting this pathway is of interest for future targeted therapies. We established novel cellular screening systems based on a STAT-responsive reporter gene (Cypridina luciferase). Of a library containing 538 microRNA (miRNA) mimics, several miRNAs affected hyper-IL-6-induced luciferase activities. When focusing on candidate miRNAs specifically targeting 3' UTRs of signaling molecules of this pathway, we identified, e.g., miR-3677-5p as a novel miRNA affecting protein expression of both STAT3 and JAK1, whereas miR-16-1-3p, miR-4473, and miR-520f-3p reduced gp130 surface expression. Interestingly, combination treatment with 2 or 3 miRNAs targeting gp130 or different signaling molecules of the pathway did not increase the inhibitory effects on phospho-STAT3 levels and STAT3 target gene expression compared to treatment with single mimics. Taken together, we identified a set of miRNAs of potential therapeutic value for cancer and inflammatory diseases, which directly target the expression of molecules within the IL-6-signaling pathway and can dampen inflammatory signal transduction., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Melanoma is an aggressive malignancy originating from pigment-producing melanocytes. The development of targeted therapies (MAPK pathway inhibitors) and immunotherapies (immune checkpoint inhibitors) led to a substantial improvement in overall survival of patients. However, the long-term efficacy of such treatments is limited by side effects, lack of clinical effects and the rapidly emerging resistance to treatment. A number of molecular mechanisms underlying this resistant phenotype have already been elucidated. In this review, we summarise currently available treatment options for metastatic melanoma and the known resistance mechanisms to targeted therapies. A focus will be placed on "phenotype switching" as a mechanism and driver of drug resistance, together with an overview of novel approaches to circumvent resistance. A large body of recent data and literature suggests that tumour progression and phenotype switching could be better controlled and development of resistance prevented or at least delayed, by combining drugs targeting fast- and slow-proliferating cells., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Melanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide. The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma patients) has markedly improved overall survival of patients with late-stage tumors, even more so when combined with MEK inhibitors targeting the same signaling pathway. However, invariably patients become resistant to this targeted therapy resulting in rapid progression with treatment-refractory disease. The purpose of this study was the identification of new kinase inhibitors that do not lead to the development of resistance in combination with BRAF inhibitors (BRAFi), or that could be of clinical benefit as a 2nd line treatment for late-stage melanoma patients that have already developed resistance., Methods: We have screened a 274-compound kinase inhibitor library in 3 BRAF mutant melanoma cell lines (each one sensitive or made resistant to 2 distinct BRAFi). The screening results were validated by dose-response studies and confirmed the killing efficacies of many kinase inhibitors. Two different tools were applied to investigate and quantify potential synergistic effects of drug combinations: the Chou-Talalay method and the Synergyfinder application. In order to exclude that resistance to the new treatments might occur at later time points, synergistic combinations were administered to fluorescently labelled parental and resistant cells over a period of > 10 weeks., Results: Eight inhibitors targeting Wee1, Checkpoint kinase 1/2, Aurora kinase, MEK, Polo-like kinase, PI3K and Focal adhesion kinase killed melanoma cells synergistically when combined with a BRAFi. Additionally, combination of a Wee1 and Chk inhibitor showed synergistic killing effects not only on sensitive cell lines, but also on intrinsically BRAFi- and treatment induced-resistant melanoma cells. First in vivo studies confirmed these observations. Interestingly, continuous treatment with several of these drugs, alone or in combination, did not lead to emergence of resistance., Conclusions: Here, we have identified new, previously unexplored (in the framework of BRAFi resistance) inhibitors that have an effect not only on sensitive but also on BRAFi-resistant cells. These promising combinations together with the new immunotherapies could be an important step towards improved 1st and 2nd line treatments for late-stage melanoma patients.

Background: Drug resistance remains an unsolved clinical issue in oncology. Despite promising initial responses obtained with BRAF and MEK kinase inhibitors, resistance to treatment develops within months in virtually all melanoma patients., Methods: Microarray analyses were performed in BRAF inhibitor-sensitive and resistant cell lines to identify changes in the transcriptome that might play a role in resistance. siRNA approaches and kinase inhibitors were used to assess the involvement of the identified Anaplastic Lymphoma Kinase (ALK) in drug resistance. The capability of extracellular vesicles (EVs) to transfer drug resistant properties was investigated in co-culture assays., Results: Here, we report a new mechanism of acquired drug resistance involving the activation of a novel truncated form of ALK. Knock down or inhibition of ALK re-sensitised resistant cells to BRAF inhibition and induced apoptosis. Interestingly, truncated ALK was also secreted into EVs and we show that EVs were the vehicle for transferring drug resistance., Conclusions: To our knowledge, this is the first report demonstrating the functional involvement of EVs in melanoma drug resistance by transporting a truncated but functional form of ALK, able to activate the MAPK signalling pathway in target cells. Combined inhibition of ALK and BRAF dramatically reduced tumour growth in vivo. These findings make ALK a promising clinical target in melanoma patients.

Interleukin (IL)-6-type cytokines have no direct antiviral activity; nevertheless, they display immune-modulatory functions. Oncostatin M (OSM), a member of the IL-6 family, has recently been shown to induce a distinct number of classical interferon stimulated genes (ISG). Most of them are involved in antigen processing and presentation. However, induction of retinoic acid-inducible gene (RIG)-I-like receptors (RLR) has not been investigated. Here we report that OSM has the capability to induce the expression of the DExD/H-Box RNA helicases RIG-I and melanoma differentiation antigen 5 (MDA5) as well as of the transcription factors interferon regulatory factor (IRF)1, IRF7 and IRF9 in primary fibroblasts. Induction of the helicases depends on tyrosine as well as serine phosphorylation of STAT1. Moreover, we could show that the OSM-induced STAT1 phosphorylation is predominantly counter-regulated by a strong STAT3-dependent SOCS3 induction, as Stat3 as well as Socs3 knock-down results in an enhanced and prolonged helicase and IRF expression. Other factors involved in regulation of STAT1 or IRF1 activity, like protein tyrosine phosphatase, non-receptor type 2 (PTPN2), promyelocytic leukaemia protein (PML) or small ubiquitin-related modifier 1 (SUMO1), play a minor role in OSM-mediated induction of RLR. Remarkably, OSM and interferon-γ (IFN-γ) synergize to mediate transcription of RLR and pre-treatment of fibroblasts with OSM fosters the type I interferon production in response to a subsequent encounter with double-stranded RNA. Together, these findings suggest that the OSM-induced JAK/STAT1 signalling is implicated in virus protection of non-professional immune cells and may cooperate with interferons to enhance RLR expression in these cells., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aberrant activation of oncogenic kinases is frequently observed in human cancers, but the underlying mechanism and resulting effects on global signaling are incompletely understood. Here, we demonstrate that the oncogenic FIP1L1-PDGFRα kinase exhibits a significantly different signaling pattern compared to its PDGFRα wild type counterpart. Interestingly, the activation of primarily membrane-based signal transduction processes (such as PI3-kinase- and MAP-kinase- pathways) is remarkably shifted toward a prominent activation of STAT factors. This diverging signaling pattern compared to classical PDGF-receptor signaling is partially coupled to the aberrant cytoplasmic localization of the oncogene, since membrane targeting of FIP1L1-PDGFRα restores activation of MAPK- and PI3K-pathways. In stark contrast to the classical cytokine-induced STAT activation process, STAT activation by FIP1L1-PDGFRα does neither require Janus kinase activity nor Src kinase activity. Furthermore, we investigated the mechanism of STAT5 activation via FIP1L1-PDGFRα in more detail and found that STAT5 activation does not involve an SH2-domain-mediated binding mechanism. We thus demonstrate that STAT5 activation occurs via a non-canonical activation mechanism in which STAT5 may be subject to a direct phosphorylation by FIP1L1-PDGFRα.

Here we describe the preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA) for the detection of STAT species. We use the method for the investigation of STAT1 and STAT3 homo- and heterodimers and show how the preparation of the extracts can influence the distribution of the STAT species observed in the EMSA. We show that detergents can massively influence the STAT dimer distribution. Although it is unclear whether they primarily interfere with STAT DNA binding and/or whether they break up or further oligomerize STATs, the observation may also have an impact on the results of other techniques performed with detergent-containing cell lysates (e.g., coimmunoprecipitations of STATs with other proteins).

Jak binding to cytokine receptors has been shown to be a complex and tight interaction. When studying loss-of-function or gain-of-function mutants of the Jaks or cytokine receptors it is often necessary to know if a certain mutant still associates correctly in the context of the signaling complex. The standard technique to show interaction of Jaks with cytokine receptors or other signalling molecules is Co-immunoprecipitation. Here we describe our protocol and discuss different pitfalls that can be encountered during the procedure.

Unlabelled: Interleukin-27 (IL-27) is a cytokine belonging to the IL-6/IL-12 cytokine family. It is secreted by antigen-presenting cells, strongly acts on T cells, and also stimulates innate immune cells. In most studies, the effects of IL-27 on T cells were investigated; however, not much is known about possible effects of IL-27 on other cell types. IL-27 signals via the common IL-6-type cytokine receptor chain gp130 and the IL-27-specific chain WSX-1. Given the importance of gp130 in regulating liver responses such as the acute phase response or liver regeneration, we investigated whether IL-27 could also have a function in liver cells. We find that IL-27 stimulates hepatoma cells and hepatocytes by inducing a sustained signal transducer and activator of transcription (STAT)1 and STAT3 activation. Whereas the STAT3 mediated responses to IL-27 (gamma-fibrinogen and hepcidin induction) are not detectable, we observe an interferon-gamma (IFN-gamma)-like STAT1 response leading to the induction of interferon-regulated proteins such as STAT1, STAT2, interferon response factor (IRF)-1, IRF-9, myxovirus resistance A and guanylate binding protein 2., Conclusion: Our study provides evidence for a function of IL-27 in hepatoma cells and hepatocytes and shows that IL-27 responses are not restricted to the classical immune cells. Our results suggest that IL-27 exerts IFN-like functions in liver cells and that it can contribute to the antiviral response in these cells.