Constitutive activation of oncogenic PDGFRα-mutant proteins occurring in GIST patients induces receptor mislocalisation and alters PDGFRα signalling characteristics.

BACKGROUND: Gastrointestinal stromal tumours (GIST) are mainly characterised by the presence of activating mutations in either of the two receptor tyrosine kinases c-KIT or platelet-derived growth factor receptor-α (PDGFRα). Most mechanistic studies dealing with GIST mutations have focused on c-KIT and far less is known about the signalling characteristics of the mutated PDGFRα proteins. Here, we study the signalling capacities and corresponding transcriptional responses of the different PDGFRα proteins under comparable genomic conditions. RESULTS: We demonstrate that the constitutive signalling via the oncogenic PDGFRα mutants favours a mislocalisation of the receptors and that this modifies the signalling characteristics of the mutated receptors. We show that signalling via the oncogenic PDGFRα mutants is not solely characterised by a constitutive activation of the conventional PDGFRα signalling pathways. In contrast to wild-type PDGFRα signal transduction, the activation of STAT factors (STAT1, STAT3 and STAT5) is an integral part of signalling mediated via mutated PDGF-receptors. Furthermore, this unconventional STAT activation by mutated PDGFRα is already initiated in the endoplasmic reticulum whereas the conventional signalling pathways rather require cell surface expression of the receptor. Finally, we demonstrate that the activation of STAT factors also translates into a biologic response as highlighted by the induction of STAT target genes. CONCLUSION: We show that the overall oncogenic response is the result of different signatures emanating from different cellular compartments. Furthermore, STAT mediated responses are an integral part of mutated PDGFRα signalling.

Interleukin-27 displays interferon-gamma-like functions in human hepatoma cells and hepatocytes.

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.

A Probabilistic Boolean Network Approach for the Analysis of Cancer-Specific Signalling: A Case Study of Deregulated PDGF Signalling in GIST.

BACKGROUND: Signal transduction networks are increasingly studied with mathematical modelling approaches while each of them is suited for a particular problem. For the contextualisation and analysis of signalling networks with steady-state protein data, we identified probabilistic Boolean network (PBN) as a promising framework which could capture quantitative changes of molecular changes at steady-state with a minimal parameterisation. RESULTS AND CONCLUSION: In our case study, we successfully applied the PBN approach to model and analyse the deregulated Platelet-Derived Growth Factor (PDGF) signalling pathway in Gastrointestinal Stromal Tumour (GIST). We experimentally determined a rich and accurate dataset of steady-state profiles of selected downstream kinases of PDGF-receptor-alpha mutants in combination with inhibitor treatments. Applying the tool optPBN, we fitted a literature-derived candidate network model to the training dataset consisting of single perturbation conditions. Model analysis suggested several important crosstalk interactions. The validity of these predictions was further investigated experimentally pointing to relevant ongoing crosstalk from PI3K to MAPK signalling in tumour cells. The refined model was evaluated with a validation dataset comprising multiple perturbation conditions. The model thereby showed excellent performance allowing to quantitatively predict the combinatorial responses from the individual treatment results in this cancer setting. The established optPBN pipeline is also widely applicable to gain a better understanding of other signalling networks at steady-state in a context-specific fashion.

The oncogenic FIP1L1-PDGFRα fusion protein displays skewed signaling properties compared to its wild-type PDGFRα counterpart.

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.

Development of an IL-6 inhibitor based on the functional analysis of murine IL-6Ralpha(1).

Dysregulated cytokine production contributes to inflammatory and proliferative diseases. Therefore, inhibition of proinflammatory mediators such as TNF, IL-1, and IL-6 is of great clinical relevance. Actual strategies are aimed at preventing receptor activation through sequestration of the ligand. Here we describe the development of an inhibitor of murine IL-6 based on fused receptor fragments. Molecular modeling-guided analysis of the murine IL-6Ralpha revealed that mutations in the Ig-like domain D1 severely affect protein function, although D1 is not directly involved in the ligand-binding interface. The resulting single chain IL-6 inhibitor (mIL-6-RFP) consisting of domains D1-D3 of mgp130, a flexible linker, and domains D1-D3 of mIL-6Ralpha is a highly potent and specific IL-6 inhibitor. mIL-6-RFP will permit further characterization of the role of IL-6 in various disease models and could ultimately lead to anti-IL-6 therapy.

Characterization of the Interleukin (IL)-6 Inhibitor IL-6-RFP: fused receptor domains act as high affinity cytokine-binding proteins.

Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6Ralpha and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFPIL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6Ralpha, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6.IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.