Systematic transcriptional profiling of responses to STAT1- and STAT3- activating cytokines in different cancer types

in Journal of Molecular Biology (2020)

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 ... [more ▼]

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- (IFN, IL-27) and STAT3-activating cytokines (IL6, OSM) were systematically compared in nine cancerous and nonneoplastic 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 up-regulated 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. [less ▲]

The PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by a-PD-L1 or a-IL6 antibodies

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 ▲]

Inflammatory signaling in liver cells: Cross-regulation of the IL-6/JAK/STAT3 pathway and microRNAs

Doctoral thesis (2017)

Interleukin-6 (IL-6) plays important roles in the regulation of liver functions and promotes the development of hepatocellular carcinoma (HCC), the most common primary liver cancer. Whereas protein ... [more ▼]

Interleukin-6 (IL-6) plays important roles in the regulation of liver functions and promotes the development of hepatocellular carcinoma (HCC), the most common primary liver cancer. Whereas protein-encoding genes are well-studied, the contribution of IL-6-regulated microRNAs (miRNAs) to its effects is largely unexplored. Similarly, little is known about miRNAs regulating key molecules of the IL-6/JAK/STAT3 signaling pathway. In the main part of this thesis, cell-based high-throughput screenings systems were developed, allowing the identification of miRNAs interfering with the IL-6/JAK/STAT3 signaling. Out of 538 miRNA mimics, this dual screening approach followed by various validation steps (luciferase-3’UTR-reporter assays, western blot and flow cytometry analyses) allowed us to identify twelve miRNAs targeting STAT3, JAK1, SOCS3, IL6R and/or gp130, ten of them had not yet been reported as regulators of this pathway before. In the second part of this work, microarray analyses revealed that primary hepatocytes are considerably more responsive to IL-6 stimulation regarding changes in their miRNomes than liver-derived cell lines. Despite their weaker response, one of the top regulated miRNA found in the primary hepatocytes, miR-146b-5p, could also be induced by IL-6-type cytokines (IL-6 and Oncostatin M) in cell lines. Its functional role(s) in hepatocytes are currently investigated. A larger, comparative study included additionally Interferon gamma and IL-27, cytokines with a STAT1-dominated response, and supplementary cell lines originating not only from the liver but also from other tissues. While all cytokines had profound effects on the mRNA transcriptomes, only very small to moderate changes were observed at the miRNome level, suggesting that the regulation mediated by cytokines mainly happens through the modulation of protein-coding genes rather than by fine-tuning via miRNAs. Additionally, we analyzed expression of a broad panel of cytokines in sera of 125 patients with liver diseases (steatosis, non-alcoholic steatohepatitis, HCC) by bead-based multiplex immunoassays. We observed that, for example, serum HGF, IL-6 and IL-8 levels increased in relation with the severeness of liver pathology while PDGF-BB and RANTES decreased. When investigating the impact of the patatin-like phospholipase domain-containing protein 3 variant I148M (PNPLA3 I148M), known to be a risk factor for the development of liver diseases, on the serum levels of cytokines, no significant differences could be observed between the different genotypes. In this PhD thesis, we identified and characterized miRNAs relevant for the IL-6/JAK/STAT3 signaling pathway, including miR-146b-5p as an IL-6-inducible miRNA. Others like miR-194-5p, miR-4473 and miR-548k were demonstrated to be negative regulators of this pathway and may be of potential therapeutic significance. In addition, we showed that, while miRNAs can modulate this pathway, IL-6 seems to rather regulate the mRNA transcriptome than the miRNome. [less ▲]