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.

Background and Aims:Metabolic dysfunction-associated steatotic liver disease (MASLD) affects >30% of adults and is becoming one of the leading causes of hepatocellular carcinoma (HCC). Reliable biomarkers are needed for the early diagnosis of HCC and detection of chronic liver diseases, like MASLD. Here we assessed the biomarker potential of circulating microRNAs in a cohort of patients genotyped for the risk allele of patatin-like phospholipase domain-containing protein 3 (PNPLA3), associated with increased susceptibility to chronic liver diseases. Methods:The cohort comprised 70 MASLD patients (40 with simple steatosis, 9 with metabolic dysfunction-associated steatohepatitis (MASH), 21 with cirrhosis), 47 HCC patients (32 with cirrhosis), and 14 healthy controls. Serum levels of miR-122-5p, miR-146a-5p, miR-146b-5p, miR-21-5p, miR-335-5p, miR-433-3p, miR-4530, miR-4651, miR-526a2, and miR-873-5p were quantified using custom qPCR plates. Their suitability for prediction of MASLD (steatosis/MASH/cirrhosis) or HCC was assessed by receiver operating characteristic curve analyses. Results:MiR-4651 and miR-21-5p were significantly reduced in sera from patients with MASLD, particularly in those with simple steatosis. Both microRNAs effectively distinguished MASLD patients with simple steatosis from healthy controls (area under the curve: 0.95 and 0.89, respectively). Moreover, miR-4651 emerged as the best predictor for differentiating "complicated" MASLD (i.e., MASH or cirrhosis) from simple steatosis; the predictive values could be increased by including additional parameters into the models (Fibroscan, thrombocytes, cytokines, or other miRNAs). miR-335-5p showed strong ability to differentiate HCC from healthy individuals (area under the curve: 0.86). The PNPLA3 p.I148M genotype was not associated with altered levels of microRNAs. Conclusion:Serum microRNAs, in particular miR-4651, may serve as additional biomarkers in patients with steatotic liver disease.

Purpose:Directing selective complement activation towards tumor cells is an attractive strategy to promote their elimination. We have generated complement-activating multimeric immunotherapeutic complexes (CoMiX), stimulating either the alternative pathway (via Factor H Related protein 4 (FHR4)) or the classical pathway (via triple Fc dimers) on HER2-expressing tumor cells. Methods:We used the C-terminal α-chain multimerizing scaffold of the C4 binding protein (C4bp) to generate CoMiX-FHR4 as well as CoMiX-Fc with 2 different anti-HER2 VHH, VHH(T) and VHH(P), recognizing trastuzumab- or pertuzumab-competing epitopes, respectively. The different CoMiX were compared in vitro for C3b and C5b9 depositions, complement-dependent cytotoxicity (CDC), and their ability to activate NK cells and phagocytosis by macrophages. We further explored their therapeutic efficacy on human BT474 tumor xenografts established in nude mice. Results:CoMiX-FHR4/VHH(T) and -FHR4/VHH(P) lead to the highest C3b and C5b9 depositions and CDC on BT474 tumor cells (p<0.0001), both individually and in combinations with their CoMiX-Fc counterparts, surpassing the low complement activating capacity of trastuzumab and pertuzumab. All CoMiX induced BT474 cell death and phagocytosis of tumor cells by macrophages while CoMiX-Fc also stimulated NK cell activation. In human BT474 xenografts sensitive to trastuzumab, CoMiX induced a massive C3b deposition 6 hours after injection. CoMiX-FHR4 reduced the tumor volume compared to controls (p< 0.05) but to a lesser extent than trastuzumab (p< 0.001) while CoMiX-VHH(P)/Fc led to a tumor volume reduction similar to pertuzumab. Combinations of two CoMiX-FHR4 or two CoMiX-Fc were more potent, similarly to the combination of trastuzumab and pertuzumab, leading to increased NK cell infiltration in xenografts. Importantly, CoMiX-FHR4 was still active against trastuzumab-resistant xenografts, delaying tumor growth and inducing a large NK cell infiltration. Conclusion:We showed here that directed complement activation on tumor cells is an alternative to therapeutic antibodies for future combination therapies upon resistance to standard-of-care treatment.

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 Directing selective complement activation towards tumor cells is an attractive strategy to promote their elimination. We have generated Complement-activating Multimeric immunotherapeutic compleXes (CoMiX) that selectively stimulate the alternative pathway using Factor H Related protein 4 (FHR4) or the classical complement pathways using triple Fc dimers on HER2-expressing tumor cells. Methods We used the C4bp C-terminal-α-/β-chain multimerising scaffolds to generate CoMiX-FHR4 and CoMiX-Fc with 2 different VHH anti-HER2, VHH(T) and VHH(P), recognising trastuzumab-or pertuzumab-competing HER2 epitopes, respectively: FHR4/VHH(T), FHR4/VHH(P), VHH(T)/Fc, VHH(P)/Fc. The different CoMiX were compared in vitro for C3b and C5b9 depositions, complement-dependent cytotoxicity, and their ability to activate NK cells and phagocytosis by macrophages using one-way ANOVA and post-hoc Tukey’s tests. We further explored their therapeutic efficacy in vivo on human BT474 breast cancer xenografts established in NUDE mice, when used individually or in combination, as compared to trastuzumab or pertuzumab. Results FHR4/VHH(T) and FHR4/VHH(P) led to the highest C3b and C5b9 depositions and CDC, both individually and in combinations on BT474 tumor cells (p< 0.0001) surpassing the very low complement activating capacity of trastuzumab and pertuzumab. CoMiX-Fc showed NK cell activation and complement-mediated BT474 phagocytosis by M2 macrophages. In the xenograft model, CoMiX-FHR4 molecules reduced the tumor volume by a factor of 7.33 compared to the PBS control. VHH(T)/Fc had no effect on tumor growth, while VHH(P)/Fc led to a 2.75-times tumor volume reduction that was higher than pertuzumab (p< 0.01). Trastuzumab and its combination with pertuzumab remained the most potent regimen, alone or in combination, to completely inhibit tumor growth. CoMiX-FHR4, CoMiX-Fc and C3b deposition were visualized as soon as one hour after injection resulting in a massive homogeneous complement deposit 6 hours after injection. Interestingly, CoMiX-FHR4 significantly reduced the growth of trastuzumab-resistant cancer cells in contrast to trastuzumab and induced a large NK cell infiltration into the tumor. Conclusions CoMiX-FHR4 and CoMiX VHH(P)/Fc significantly inhibit tumor growth through complement activation, NK cells infiltration, and phagocytosis by macrophages. CoMiX-FHR4 proteins delay xenograft growth of BT474 cells resistant to trastuzumab and could thus be an attractive approach when resistance to antibody emerges. What is already known on this topic Complement activation represents a substantial part of the overall biological activity of few therapeutic antibodies used in cancer immunotherapy. Factor H-related protein 4 can activate complement by serving as a platform for the assembly of alternative pathway C3 convertase by competing with factor H for C3b binding. We previously showed that multimeric recombinant proteins displaying the FHR4 complement effector moiety and a nanobody anti-HER2 targeting moiety selectively direct the activation of the complement alternative pathway on HER2-expressing tumor cells, leading to subsequent cell destruction through direct cell lysis or through the activation of host effector cells. What this study adds We used in the current work a novel complement-directed tumor cell distruction strategy in vivo . We showed that CoMiX-FHR4 and CoMiX-Fc (based on triple Fc dimers), targeting HER2-positive breast tumor cells, inhibit tumor growth in a model of BT474 xenograft in NUDE mice by stimulating complement activation, BT474 death, NK cell activation, and phagocytosis of tumor cells by macrophages. CoMiX-FHR4 remain efficient in xenografts of BT474 cells resistant to trastuzumab. How this study might affect research, practice or policy We demonstrate for the first time that directed complement activation on tumor cells is an alternative to therapeutic antibodies which is particularly promising when resistance to standard-of-care treatment occurs. ### Competing Interest Statement A patent application has been granted in USA for CoMiX (WO2017202776) by the inventors (C.S.D, J.H.M.C, X.D). The authors have declared that no other conflict of interest exists.

Supplementary Table and Figure Legends, Material and Methods from Signatures of MicroRNAs and Selected MicroRNA Target Genes in Human Melanoma

Supplementary Figure 2 from Signatures of MicroRNAs and Selected MicroRNA Target Genes in Human Melanoma

BACKGROUND:Individuals carrying the risk variant p.I148M of patatin-like phospholipase domain-containing protein 3 (PNPLA3) have a higher susceptibility to fatty liver diseases and associated complications, including HCC, a cancer closely linked to chronic inflammation. Here, we assessed circulating cytokine profiles for patients with chronic liver diseases genotyped for PNPLA3. METHODS:Serum concentrations of 22 cytokines were measured by multiplex sandwich-ELISA. The cohort comprised 123 individuals: 67 patients with NAFLD without cirrhosis (57 steatosis, 10 NASH), 24 patients with NAFLD with cirrhosis, 21 patients with HCC (15 cirrhosis), and 11 healthy controls. Receiver operator characteristic analyses were performed to assess the suitability of the cytokine profiles for the prediction of steatosis, cirrhosis, and HCC. RESULTS:HGF, IL-6, and IL-8 levels were increased in patients, with ∼2-fold higher levels in patients with cirrhosis versus healthy, while platelet derived growth factor-BB (PDGF-BB) and regulated on activation, normal T cell expressed and secreted (RANTES) showed lower concentrations compared to controls. Migration inhibitory factor and monocyte chemoattractant protein-1 (MCP-1) were found at higher levels in NAFLD samples (maximum: NAFLD-cirrhosis) versus healthy controls and HCC samples. In receiver operator characteristic analyses, migration inhibitory factor, IL-8, IL-6, and monocyte chemoattractant protein-1 yielded high sensitivity scores for predicting noncirrhotic NAFLD (vs. healthy). The top combination to predict cirrhosis was HGF plus PDGF-BB. Migration inhibitory factor performed best to discriminate HCC from NAFLD; the addition of monokine induced gamma (MIG), RANTES, IL-4, macrophage colony-stimulating factor (M-CSF), or IL-17A as second parameters further increased the AUC values (> 0.9). No significant impact of the PNPLA3I148M allele on cytokine levels was observed in this cohort. CONCLUSIONS:Cytokines have biomarker potential in patients with fatty liver, possibly suited for early HCC detection in patients with fatty liver. Patients carrying the PNPLA3 risk allele did not present significantly different levels of circulating cytokines.

Supplementary Figure 1 from Signatures of MicroRNAs and Selected MicroRNA Target Genes in Human Melanoma

Supplementary Figures S1-S4 from Constitutive Suppressor of Cytokine Signaling 3 Expression Confers a Growth Advantage to a Human Melanoma Cell Line

Hypoxia is a common hallmark of solid tumors and is associated with aggressiveness, metastasis and poor outcome. Cancer cells under hypoxia undergo changes in metabolism and there is an intense crosstalk between cancer cells and cells from the tumor microenvironment. This crosstalk is facilitated by small extracellular vesicles (sEVs; diameter between 30 and 200 nm), including exosomes and microvesicles, which carry a cargo of proteins, mRNA, ncRNA and other biological molecules. Hypoxia is known to increase secretion of sEVs and has an impact on the composition of the cargo. This sEV-mediated crosstalk ultimately leads to various biological effects in the proximal tumor microenvironment but also at distant, future metastatic sites. In this review, we discuss the changes induced by hypoxia on sEV secretion and their cargo as well as their effects on the behavior and metabolism of cancer cells, the tumor microenvironment and metastatic events.

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.

Reduced levels of intratumoural oxygen are associated with hypoxia-induced pro-oncogenic events such as invasion, metabolic reprogramming, epithelial-mesenchymal transition, metastasis and resistance to therapy, all favouring cancer progression. Small extracellular vesicles (EV) shuttle various cargos (proteins, miRNAs, DNA and others). Tumour-derived EVs can be taken up by neighbouring or distant cells in the tumour microenvironment, thus facilitating intercellular communication. The quantity of extracellular vesicle secretion and their composition can vary with changing microenvironmental conditions and disease states. Here, we investigated in melanoma cells the influence of hypoxia on the content and number of secreted EVs. Whole miRNome and proteome profiling revealed distinct expression patterns in normoxic or hypoxic growth conditions. Apart from the well-known miR-210, we identified miR-1290 as a novel hypoxia-associated microRNA, which was highly abundant in hypoxic EVs. On the other hand, miR-23a-5p and -23b-5p were consistently downregulated in hypoxic conditions, while the protein levels of the miR-23a/b-5p-predicted target IPO11 were concomitantly upregulated. Furthermore, hypoxic melanoma EVs exhibit a signature consisting of six proteins (AKR7A2, DDX39B, EIF3C, FARSA, PRMT5, VARS), which were significantly associated with a poor prognosis for melanoma patients, indicating that proteins and/or miRNAs secreted by cancer cells may be exploited as biomarkers.

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.

OBJECTIVE:Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. METHODS:miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. RESULTS:We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid β-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. CONCLUSION:GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment.

1University of Luxembourg, Life Sciences ResearchUnit—Signal Transduction Laboratory, Belvaux, Luxembourg 2University of Luxembourg, Life Sciences ResearchUnit—Bioinformatics Core Facility, Belvaux, Luxembourg 3UniversityHospitalWürzburg,Medical Clinic II, Division ofHepatology,Würzburg, Germany 4Proteome andGenomeResearchUnit, Department ofOncology, Luxembourg Institute of Health, Luxembourg, Luxembourg 5University of Luxembourg, Life Sciences ResearchUnit—MolecularDiseaseMechanisms Laboratory, Belvaux, Luxembourg Correspondence ClaudeHaan,University of Luxembourg, Life SciencesResearchUnit—Signal Transduction Laboratory, 6, avenueduSwing, L-4367Belvaux, Luxembourg. E-mail: claude.haan@uni.lu Abstract 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 observedwithin the different cell lines andwhen 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.

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.

Interleukin-6 (IL-6)-type cytokines play important roles in liver (patho-)biology. For instance, they regulate the acute phase response to inflammatory signals and are involved in hepatocarcinogenesis. Much is known about the regulation of protein-coding genes by cytokines whereas their effects on the miRNome is less well understood. We performed a microarray screen to identify microRNAs (miRNAs) in human hepatocytes which are modulated by IL-6-type cytokines. Using samples of 2 donors, 27 and 68 miRNAs (out of 1,733) were found to be differentially expressed upon stimulation with hyper-IL-6 (HIL-6) for up to 72 h, with an overlap of 15 commonly regulated miRNAs. qPCR validation revealed that miR-146b-5p was also consistently up-regulated in hepatocytes derived from 2 other donors. Interestingly, miR-146b-5p (but not miR-146a-5p) was induced by IL-6-type cytokines (HIL-6 and OSM) in non-transformed liver-derived PH5CH8 and THLE2 cells and in Huh-7 hepatoma cells, but not in HepG2 or Hep3B hepatoma cells. We did not find evidence for a differential regulation of miR-146b-5p expression by promoter methylation, also when analyzing the TCGA data set on liver cancer samples. Inducible overexpression of miR-146b-5p in PH5CH8 cells followed by RNA-Seq analysis revealed effects on multiple mRNAs, including those encoding IRAK1 and TRAF6 crucial for Toll-like receptor signaling. Indeed, LPS-mediated signaling was attenuated upon overexpression of miR-146b-5p, suggesting a regulatory loop to modulate inflammatory signaling in hepatocytes. Further validation experiments suggest DNAJC6, MAGEE1, MPHOSPH6, PPP2R1B, SLC10A3, SNRNP27, and TIMM17B to be novel targets for miR-146b-5p (and miR-146a-5p).