In a compound screen on melanoma cells, we identified FX-11 as one of the compounds inhibiting the growth of sensitive and Encorafenib/Binimetinib-resistant 624Mel and Wm3248 melanoma cells. FX-11, reported to be a lactate dehydrogenase inhibitor (LDHi), did not decrease the NAD+/NADH ratio, glucose uptake, or lactate secretion of melanoma cells, unlike other LDH inhibitors we used as controls. Instead, FX-11 increased both the oxygen consumption and the extracellular acidification rate of cells in Seahorse assays, behaving similarly to the mitochondrial uncouplers FCCP and BAM15. FX-11 could also be used as an uncoupler in Mito Stress tests. In addition, FX-11 and BAM15 drastically decreased the mitochondrial membrane potential, in contrast to the LDH inhibitors LDH-IN-I and GNE-140. Finally, FX-11 treatment induced an increase in AMPK and acetyl-CoA carboxylase (ACC) phosphorylation and a decrease in phosphorylation of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), indicative of AMPK activation by decreased ATP/AMP ratio. The observed AMPK activation seems to be involved in FX-11-mediated cell death since knock-down of AMPKα1 reduced cell death. Specific activation of AMPK by MK8722, on the other hand, increased cell death.Taken together, we provide evidence that FX-11 inhibits the growth of melanoma cells, including drug-resistant ones, through an AMPK-dependent mechanism by acting as a mitochondrial uncoupler. Our data do not support that FX-11 acts as an LDH inhibitor.

Resistance of melanoma cells to targeted therapy (BRAF and MEK inhibitors) is a major clinical problem and alternative treatments are sought. We describe the establishment of modular physiologic medium (MPM) and Mel-MPM (which contains additional supplements and sustains the 3D growth of melanoma cells, fibroblasts (NHDFs) and HMEC-1 endothelial cells) as novel resources for melanoma and combine them with a multi-cell-type matrix-embedded 3D culture model to investigate melanoma cell vulnerabilities in a more physiological setting. We made use of the modular nature of MPM to interrogate NEAA dependencies in melanoma cells and we found them to be particularly sensitive to the depletion of C/C. We additionally describe that melanoma cells are less sensitive to ferroptosis inducing compounds when cultured in MPM compared to RPMI and we could attribute this to different components of MPM and Mel-MPM (selenite, B27). Cell death induced by the glutathione peroxidase 4 inhibitor, ML162, had characteristics of ferroptosis or apoptosis depending on cell type, its drug resistance status and the culture medium. Cystine/cysteine starvation and ML162 treatment combinations increased melanoma cell death in 2D, 3D, and also in the complex matrix embedded multi-cell-type-3D system in Organoplates. This underlines the potential of combining metabolism-oriented drug treatments with amino acid starvation conditions, which is of interest in view of future therapeutic approaches to combat melanoma and other cancer types. ### Competing Interest Statement The authors have declared no competing interest.

Many melanoma patients do not respond to therapy or rapidly develop resistance to MAPK pathway inhibitors and immune checkpoint blockade treatments, highlighting the urgent need for additional therapeutic strategies for these patients. To identify compounds that target drug-naive and -resistant melanoma cells (Encorafenib/Binimetinib-resistant), we performed a screen using a metabolism-oriented compound library in different cell culture media and growth conditions (2D and 3D). The efficacy of several compounds varied considerably under changing test conditions, but importantly, we also detected compounds that work across all tested conditions. In general, drug activity was reduced under hypoxia and in 3D spheroids. Thorough validation was performed for drugs showing potency in all conditions with a focus on efficacy in 3D spheroids grown in an in-house physiological culture medium. Using hydrogel matrix-embedded multi-cell type 3D models, we found that FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, is very potently suppressing melanoma cell growth while not affecting the growth of healthy cells such as fibroblasts and endothelial cells. ### Competing Interest Statement The authors have declared no competing interest.

Background and aims: It was found that secretory SCUBE3 protein is highly expressed in lung cancer and osteosarcoma, is associated with proliferation, invasion and metastasis of lung cancer cells and breast cancer cells.Therefore, the aim is to explore the clinical significance of SCUBE3 in HCC and the specific molecular mechanism of SCUBE3 on the biological function.Method: TCGA database and IHC were used to detect the SCUBE3 of cancer and paracancerous.CCK8, Clone formation and celigo experiment were used to detect the regulation of SCUBE3 on the proliferation of hepatoma cells.The apoptosis and cell cycle after knockdown of SCUBE3 were detected by flow cytometry.Cell migration and invasion were detected by cell scratch and Transwell.The invasion and EMT markers were detected by WB and qPCR.EMT markers in nude mouse tumours was detected by IHC.The cell movement was observed by the RTCA system.ROS reactive oxygen species assay was used to evaluate the effect of SCUBE3 on cell adhesion.The xenograft tumour and metastasis model of liver cancer were constructed.The interacting proteins were verified by confocal and IP assay.TGF β was added on the basis of knocking down SCUBE3.SCUBE3 recombinant protein induced EMT formation.The interaction between Sox9 and SCUBE3 was verified by Chip experiment.Results: SCUBE3 in hepatocellular carcinoma was different from that in adjacent tissues ( p = 0.017), and SCUBE3 was related to age and stage and grade of hepatocellular carcinoma.SCUBE3 knockdown inhibit the HCC cell proliferation and colony formation, migration and invasion.The apoptosis increased and the transformation of the cell cycle from the G1 phase to S phase inhibited in KD group.The degree of ROS in different cells was different.The lung metastasis model of liver cancer evaluated the regulatory ability of SCUBE3 on the invasion and metastasis of liver cancer cells.The overexpression of metastatic tumors in the upper lung group was more than that in the control group.Akt and TGFβ were the pathways related to the regulation of hepatoma cell proliferation, metastasis and EMT β/Smad2/3 pathway.SCUBE3 and TGFβ R II has interaction.Smad2/3 pathway was also inhibited in KD group.VEGF, N-cadherin also increased, E-cadherin decreased and Smad2/3 expression increased when TGF β was given in KD group.Sox9 and Runx1 were inhibited with SCUBE3 knockdown.The function of Sox9 was related to the prognosis of liver cancer.Chip experiment showed that there was SCUBE3 binding site in chromosome 1657-1665 region of Sox9 transcription factor.Conclusion: TGF-β-Sox9-SCUBE3 axis regulates the Akt pathway and EMT to promote the proliferation, invasion and metastasis of HCC cells.SCUBE3 may become a new target for clinical diagnosis and treatment of HCC.

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.

Cytokines orchestrate responses to pathogens and in inflammatory processes, but they also play an important role in cancer by shaping the expression levels of cytokine response genes. Here, we conducted a large profiling study comparing miRNome and mRNA transcriptome data generated following different cytokine stimulations. Transcriptomic responses to STAT1- (IFNγ, IL-27) and STAT3-activating cytokines (IL6, OSM) were systematically compared in nine cancerous and non-neoplastic cell lines of different tissue origins (skin, liver and colon). The largest variation in our datasets was seen between cell lines of the three different tissues rather than stimuli. Notably, the variability in miRNome datasets was a lot more pronounced than in mRNA data. Our data also revealed that cells of skin, liver and colon tissues respond very differently to cytokines and that the cell signaling networks activated or silenced in response to STAT1- or STAT3-activating cytokines are specific to the tissue and the type of cytokine. However, globally, STAT1-activating cytokines had stronger effects than STAT3-inducing cytokines with most significant responses in liver cells, showing more genes upregulated and with higher fold change. A more detailed analysis of gene regulations upon cytokine stimulation in these cells provided insights into STAT1- versus STAT3-driven processes in hepatocarcinogenesis. Finally, independent component analysis revealed interconnected transcriptional networks distinct between cancer cells and their healthy counterparts.

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.

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.

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. 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. 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. 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.

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--like response in the microarray analyses, with up-regulation of genes involved in antiviral defense, antigen presentation, and immune suppression. Like IFN-, 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 associated with immune escape of cancer. Interestingly, differential expression of these genes was observed within the different cell lines and when comparing IL27 to IFN-. 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 T(H)1 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 pre-stimulationmimicking a situation occurring, for example, in IL6-secreting tumors or in tumor inflammation-induced cachexiacan 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 IL27 with blocking antibodies against PD-L1 or/and IL6-type cytokines.

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.

The constitutively active Janus kinase 2 mutant Jak2-V617F is responsible for cytokine-independent growth of hematopoietic cells and the development of myeloproliferative neoplasms, such as polycythaemia vera and essential thrombocythaemia. Cells expressing Jak2-V617F exhibit constitutive STAT, MAPK, and PI3K signalling, and constitutive association of the multi-site docking protein Gab1 to PIP3 at the plasma membrane. Here, we demonstrate the crucial role of Gab1 for the proliferation of Jak2-V617F-positive human erythroleukaemia (HEL) cells. In Jak2-V617F-expressing cells Gab1 is constitutively phosphorylated by Erk1/2 on serine residue 552, which regulates binding to PIP3. Additionally, Gab1 is constitutively phosphorylated on tyrosine residue 627. Tyrosine 627 is a SHP2 binding site and required for Gab1-dependent Erk1/2 activation. As previously shown, Jak2-V617F-dependent Erk1/2 and PI3K activation act synergistically on the proliferation of Jak2-V617F-positive cells. Here, we examined whether constitutive membrane association of Gab1 explains cytokine-independent Gab1 phosphorylation in Jak2-V617F-expressing cells. Although we could demonstrate Jak2-V617F-dependent constitutive serine 552 and tyrosine 627 phosphorylation of Gab1, interestingly, both phosphorylations do not require binding of Gab1 to PIP3 at the plasma membrane. Instead, we observed a constitutive interaction of Gab1 with the erythropoietin receptor in Jak2-V617F-expressing cells, which depends on Janus kinase activity. Thus, constitutive Gab1-dependent signalling in Jak2-V617F-expressing cells does not occur due to the constitutive association of Gab1 with PIP3 at the plasma membrane.

Interleukin-27 (IL27) is a type-I-cytokine of the IL6/IL12 family predominantly secreted by activated macrophages and dendritic cells. In the liver, IL27 expression was observed to be upregulated in patients with hepatitis B, and sera of hepatocellular carcinoma (HCC) patients contain significantly elevated levels of IL27 compared to healthy controls or patients with hepatitis and/or liver cirrhosis. In this study, we show that IL27 induces STAT1 and STAT3 phosphorylation in 5 HCC lines and 3 different types of non-transformed liver cells. We were especially interested in the relevance of the IL27-induced STAT3 activation in liver cells. Thus, we compared the IL27 responses with those induced by IFNγ (STAT1-dominated response) or IL6-type cytokines (IL6, hyper-IL6 (hy-IL6) or OSM) (STAT3-dominated response) by microarray analysis and find that in HCC cells, IL27 induces an IFNγ-like, STAT1-dependent transcriptional response, but we do not find an effective STAT3-dependent response. Validation experiments corroborate the finding from the microarray evaluation. Interestingly, the availability of STAT1 seems critical in the shaping of the IL27 response, as the siRNA knock-down of STAT1 revealed the ability of IL27 to induce the acute-phase protein γ-fibrinogen, a typical IL6 family characteristic. Moreover, we describe a crosstalk between the signaling of IL6-type cytokines and IL27: responses to the gp130-engaging cytokine IL27 (but not those to IFNs) can be inhibited by IL6-type cytokine pre-stimulation, likely by a SOCS3-mediated mechanism. Thus, IL27 recapitulates IFNγ responses in liver cells, but differs from IFNγ by its sensitivity to SOCS3 inhibition.

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.

Background Most melanoma patients with BRAF V600E 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 BRAF V600E has been shown to affect the metabolism. Methods Time course experiments and a series of western blots were performed in a panel of BRAF V600E and BRAF WT /NRAS mut 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 BRAF V600E and in BRAF WT /NRAS mut 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 BRAF V600E and BRAF WT /NRAS mut 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% NRAS mut , 11.9% NF1 mut ).

The pyruvate dehydrogenase complex (PDC) is an important gatekeeper enzyme connecting glycolysis to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Thereby, it has a strong impact on the glycolytic flux as well as the metabolic phenotype of a cell. PDC activity is regulated via reversible phosphorylation of three serine residues on the pyruvate dehydrogenase (PDH) E1α subunit. Phosphorylation of any of these residues by the PDH kinases (PDKs) leads to a strong decrease in PDC activity. Under hypoxia, the inactivation of the PDC has been described to be dependent on the hypoxia-inducible factor 1 (HIF-1)-induced PDK1 protein upregulation. In this study, we show in two hepatocellular carcinoma cell lines (HepG2 and JHH-4) that, during the adaptation to hypoxia, PDH is already phosphorylated at time points preceding HIF-1-mediated transcriptional events and PDK1 protein upregulation. Using siRNAs and small molecule inhibitor approaches, we show that this inactivation of PDC is independent of HIF-1α expression but that the PDKs need to be expressed and active. Furthermore, we show that reactive oxygen species might be important for the induction of this PDH phosphorylation since it correlates with the appearance of an altered redox state in the mitochondria and is also inducible by H2O2 treatment under normoxic conditions. Overall, these results show that neither HIF-1 expression nor PDK1 upregulation is necessary for the phosphorylation of PDH during the first hours of the adaptation to hypoxia.

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 α (HIF-1 α) under normoxic conditions in hepatocytes and hepatoma cells. HIF-1 α 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 α 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 α 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.

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.