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See detailThe PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by a-PD-L1 or a-IL6 antibodies
Rolvering, Catherine UL; Zimmer, Andreas David UL; Ginolhac, Aurélien UL et al

in Journal of Leukocyte Biology (2018), 104

Interleukin-27 (IL27) is a type-I cytokine of the IL6/IL12 family and is predominantly secreted by activated macrophages and dendritic cells.We show that IL27 induces STAT factor phosphorylation in ... [more ▼]

Interleukin-27 (IL27) is a type-I cytokine of the IL6/IL12 family and is predominantly secreted by activated macrophages and dendritic cells.We show that IL27 induces STAT factor phosphorylation in cancerous cell lines of different tissue origin. IL27 leads to STAT1 phosphorylation and recapitulates an IFN-𝛾-like response in the microarray analyses, with up-regulation of genes involved in antiviral defense, antigen presentation, and immune suppression. Like IFN-𝛾, IL27 leads to an up-regulation of TAP2 and MHC-I proteins, which mediate increased tumor immune clearance. However, both cytokines also upregulate proteins such as PD-L1 (CD274) and IDO-1, which are associatedwith immune escape of cancer. Interestingly, differential expression of these geneswas observed within the different cell lines and when comparing IL27 to IFN-𝛾. In coculture experiments of hepatocellular carcinoma (HCC) cells with peripheral blood mononuclear cells, pre-treatment of the HCC cells with IL27 resulted in lowered IL2 production by anti-CD3/-CD28 activated T-lymphocytes. Addition of anti-PD-L1 antibody, however, restored IL2 secretion. The levels of other TH1 cytokines were also enhanced or restored upon administration of anti-PD-L1. In addition, we show that the suppression of IL27 signaling by IL6-type cytokine prestimulation— mimicking a situation occurring, for example, in IL6-secreting tumors or in tumor inflammation–induced cachexia—can be antagonized by antibodies against IL6-type cytokines or their receptors. Therapeutically, the antitumor effects of IL27 (mediated, e.g., by increased antigen presentation) might thus be increased by combining IL27with blocking antibodies against PD-L1 or/and IL6-type cytokines. [less ▲]

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See detailROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells
Cesi, Giulia UL; Walbrecq, Geoffroy UL; Zimmer, Andreas David UL et al

in Molecular Cancer (2017), 8(june),

Background: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of ... [more ▼]

Background: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism. Methods: Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/ NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results: We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. Conclusions: In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut). [less ▲]

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See detailThe role of HIF-1 in oncostatin M-dependent metabolic reprogramming of hepatic cells.
Battello, Nadia UL; Zimmer, Andreas David UL; Goebel, Carole et al

in Cancer & metabolism (2016), 4

BACKGROUND: Hypoxia and inflammation have been identified as hallmarks of cancer. A majority of hepatocellular carcinomas are preceded by hepatitis B- or C-related chronic infections suggesting that liver ... [more ▼]

BACKGROUND: Hypoxia and inflammation have been identified as hallmarks of cancer. A majority of hepatocellular carcinomas are preceded by hepatitis B- or C-related chronic infections suggesting that liver cancer development is promoted by an inflammatory microenvironment. The inflammatory cytokine oncostatin M (OSM) was shown to induce the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) under normoxic conditions in hepatocytes and hepatoma cells. HIF-1 alpha is known to orchestrate the expression of numerous genes, many of which code for metabolic enzymes that play key roles in the adaptation of cellular metabolism to low oxygen tension. RESULTS: Here, we show that OSM-induced upregulation of HIF-1 alpha reprograms cellular metabolism in three clones of the human hepatocyte cell line PH5CH (PH5CH1, PH5CH7, and PH5CH8) towards a hypoxia-like metabolic phenotype but has no significant effect on cellular metabolism of HepG2 and JHH-4 hepatoma cells. Although we observed only minor changes in glucose uptake and lactate secretion in PH5CH8 upon OSM treatment, we identified more pronounced changes in intracellular fluxes based on stable isotope labeling experiments. In particular, glucose oxidation in the tricarboxylic acid (TCA) cycle is reduced through pyruvate dehydrogenase kinase 1 (PDK1)-mediated inhibition of the pyruvate dehydrogenase complex, thereby reducing the oxidative TCA cycle flux. As a result of the impaired mitochondrial glucose and glutamine oxidation, the reductive isocitrate dehydrogenase flux was increased. CONCLUSIONS: We provide evidence that connects the inflammatory mediator OSM to a hypoxia-like metabolic phenotype. In the human hepatocyte cell line PH5CH, OSM-mediated upregulation of HIF-1 alpha and PDK1 can induce hypoxia-like metabolic changes, although to a lesser extent than hypoxia itself. Since PDK1 is overexpressed in several cancers, it might provide a causal link between chronic inflammation and malignant cellular transformation. [less ▲]

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See detailCyclooxygenase-2 contributes to the selective induction of cell death by the endocannabinoid 2-arachidonoyl glycerol in hepatic stellate cells.
Siegmund, S. V.; Wojtalla, A.; Schlosser, M. et al

in Biochemical and biophysical research communications (2016), 470(3), 678-84

The endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is an anti-fibrotic lipid mediator that induces apoptosis in hepatic stellate cells (HSCs), but not in hepatocytes. However, the exact molecular ... [more ▼]

The endogenous cannabinoid 2-arachidonoyl glycerol (2-AG) is an anti-fibrotic lipid mediator that induces apoptosis in hepatic stellate cells (HSCs), but not in hepatocytes. However, the exact molecular mechanisms of this selective induction of HSC death are still unresolved. Interestingly, the inducible isoform of cyclooxygenase, COX-2, can metabolize 2-AG to pro-apoptotic prostaglandin glycerol esters (PG-GEs). We analyzed the roles of COX-2 and endocannabinoid-derived PG-GEs in the differential susceptibility of primary activated HSCs and hepatocytes toward 2-AG-induced cell death. HSCs displayed significant COX-2 expression in contrast to hepatocytes. Similar to 2-AG, treatment of HSCs with PGD2-GE dose-dependently induced cell death independently from cannabinoid receptors that was accompanied by PARP- and caspase 3-cleavage. In contrast to 2-AG, PGD2-GE failed to induce significant ROS formation in HSCs, and depletion of membrane cholesterol did not rescue HSCs from PGD2-GE-induced apoptosis. These findings indicate differential engagement of initial intracellular signaling pathways by 2-AG and its COX-2-derived metabolite PGD2-GE, but similar final cell death pathways. Other PG-GEs, such as PGE2-or PGF2alpha-GE did not induce apoptosis in HSCs. Primary rat hepatocytes were mainly resistant against 2-AG- and PGD2-GE-induced apoptosis. HSCs, but not hepatocytes were able to metabolize 2-AG to PGD2-GE. As a proof of principle, HSCs from COX-2(-/-) mice lacked PDG2-GE production after 2-AG treatment. Accordingly, COX-2(-/-) HSCs were resistant against 2-AG-induced apoptosis. In conclusion, the divergent expression of COX-2 in HSCs and hepatocytes contributes to the different susceptibility of these cell types towards 2-AG-induced cell death due to the generation of pro-apoptotic PGD2-GE by COX-2 in HSCs. Modulation of COX-2-driven metabolization of 2-AG may provide a novel physiological concept allowing the specific targeting of HSCs in liver fibrosis. [less ▲]

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See detailDIFFERENTIAL EFFECTS OF IL-6-TYPE CYTOKINES, THEIR INTERPLAY WITH HYPOXIA AND RESULTING EFFECTS ON THE METABOLISM
Zimmer, Andreas David UL

Doctoral thesis (2015)

IL-6-type cytokines signal mainly via the Jak/STAT pathway using the common receptor chain gp130. They are implicated in various biological processes such as differentiation, apoptosis, tissue ... [more ▼]

IL-6-type cytokines signal mainly via the Jak/STAT pathway using the common receptor chain gp130. They are implicated in various biological processes such as differentiation, apoptosis, tissue regeneration and proliferation. However, one of their main functions is the regulation of inflammatory processes, especially the initial induction of an inflammatory response. They hereby exert key steps in the onset as well as in the termination of an inflammation and in the promotion of the shift from the innate to the adaptive immune response. In hepatocellular carcinoma (HCC) IL-6 has been identified as a key factor in the onset as well as in the progression of this cancer. In this study we first investigated the effects of the IL-6-type cytokine IL-27 on hepatic cells. By evaluating IL-27 signalling in several HCC cell lines as well as hepatic cells, we could demonstrate that IL-27 does, in contrary to other IL-6-type cytokines, activate STAT1 and STAT3. A transcriptional and protein read-out of STAT3 was not found in hepatic cells. Thus, IL-27 exerts interferon γ-like functions only in the liver. Interestingly, we could further show that the IL-27 signalling can still be repressed by the canonical IL-6-type cytokine feedback regulator SOCS3, following a pre-stimulation with other IL-6-type cytokines. The second part of this thesis focused on the effects of the IL-6-type cytokine Oncostatin M (OSM) on the cellular metabolism. We hereby evaluated to which extent OSM-mediated STAT3 activation can induce a Warburg-like, highly glycolytic, metabolic phenotype under normoxia. OSM induced the expression of HIF-1α, a key factor mediating the shift to a highly glycolytic phenotype, in several HCC cell lines and immortalized hepatocytes. In the non-neoplastic hepatocyte cell line, PH5CH8, this led to the induction of a more glycolytic metabolic phenotype. The observed changes in these immortalized hepatocytes did not fully resemble the metabolic state seen under hypoxia, since mainly the observed induction of PDK1 seems to explain the effect. Overall we could not observe strong effects on glycolytic enzymes or other canonical HIF-1α target genes involved in metabolism, on a transcriptional or protein expression level in the investigated cells. Interestingly we found that HIF-1α does not seem to be mediating all the early adaptions of the cellular metabolism to hypoxia. The early inhibition of the pyruvate dehydrogenase complex could be shown to be independent of the HIF-1α-mediated up-regulation of the pyruvate dehydrogenase kinase 1, but might rather be induced by reactive oxygen species, created during the switch from normoxia to hypoxia. In summary we could show that in the liver IL-27 exerts interferon γ-like effects. Additionally, cytokine-activated STAT3 does not per se induce a glycolytic metabolic phenotype and HIF-1α is not necessary for some of the early adaptions of the cellular metabolism to hypoxia. [less ▲]

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See detailCK2 phosphorylation of C/EBPdelta regulates its transcription factor activity.
Schwind, Lisa; Zimmer, Andreas David UL; Gotz, Claudia et al

in The international journal of biochemistry & cell biology (2015), 61

Protein kinase CK2 plays an essential role in cell viability in lower and higher eukaryotes. As a global regulator it phosphorylates and thereby regulates a broad array of cellular targets including a ... [more ▼]

Protein kinase CK2 plays an essential role in cell viability in lower and higher eukaryotes. As a global regulator it phosphorylates and thereby regulates a broad array of cellular targets including a large number of transcription factors. Here, we have identified the CCAAT/enhancer binding protein delta (C/EBPdelta) as a new substrate for CK2. Using point mutants of C/EBPdelta the major phosphorylation site for CK2 was mapped to serine 57, which is located within the transactivation domain of C/EBPdelta. For proper functioning as a transcription factor C/EBPdelta has to be translocated into the nucleus where it forms heterodimers with other members of the C/EBP family of proteins and ATF4. Here, we found that CK2 phosphorylation does neither influence the subcellular localization of C/EBPdelta nor its interaction with C/EBPbeta, but rather does CK2 phosphorylation modulate the transcriptional activity of C/EBPdelta. Moreover, we found that CK2 bound to C/EBPdelta, which might help to target CK2 to the transcriptional machinery where it can phosphorylate other transcription factors or co-activators. [less ▲]

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See detailSequence interpretation. Functional annotation of mouse genome sequences.
Nadeau, J. H.; Balling, Rudi UL; Barsh, G. et al

in Science (2001), 291(5507), 1251-5

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See detailGenome-wide, large-scale production of mutant mice by ENU mutagenesis.
Hrabe de Angelis, M. H.; Flaswinkel, H.; Fuchs, H. et al

in Nature Genetics (2000), 25(4), 444-7

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model ... [more ▼]

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html). [less ▲]

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