Transcriptional variations in the wider peritumoral tissue environment of pancreatic cancer

in International Journal of Cancer (2018), 142(5), 10101021

Transcriptional profiling was performed on 452 RNA preparations isolated from various types of pancreatic tissue from tumour patients and healthy donors, with a particular focus on peritumoral samples ... [more ▼]

Transcriptional profiling was performed on 452 RNA preparations isolated from various types of pancreatic tissue from tumour patients and healthy donors, with a particular focus on peritumoral samples. Pancreatic ductal adenocarcinomas (PDAC) and cystic tumours were most different in these non-tumorous tissues surrounding them, whereas the actual tumours exhibited rather similar transcript patterns. The environment of cystic tumours was transcriptionally nearly identical to normal pancreas tissue. In contrast, the tissue around PDAC behaved a lot like the tumour, indicating some kind of field defect, while showing far less molecular resemblance to both chronic pancreatitis and healthy tissue. This suggests that the major pathogenic difference between cystic and ductal tumours may be due to their cellular environment rather than the few variations between the tumours. Lack of correlation between DNA methylation and transcript levels makes it unlikely that the observed field defect in the peritumoral tissue of PDAC is controlled to a large extent by such epigenetic regulation. Functionally, a strikingly large number of autophagy-related transcripts was changed in both PDAC and its peritumoral tissue, but not in other pancreatic tumours. A transcription signature of 15 autophagy-related genes was established that permits a prognosis of survival with high accuracy and indicates the role of autophagy in tumour biology. [less ▲]

Crosstalk between different family members: IL27 recapitulates IFNγ responses in HCC cells, but is inhibited by IL6-type cytokines

in BBA Molecular Cell Research (2017)

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 ... [more ▼]

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. [less ▲]

Gene Regulatory Network Inference of Immunoresponsive Gene 1 (IRG1) Identifies Interferon Regulatory Factor 1 (IRF1) as Its Transcriptional Regulator in Mammalian Macrophages

in PLoS ONE (2016)

Immunoresponsive gene 1 (IRG1) is one of the highest induced genes in macrophages under pro-inflammatory conditions. Its function has been recently described: it codes for immune-responsive gene 1 protein ... [more ▼]

Immunoresponsive gene 1 (IRG1) is one of the highest induced genes in macrophages under pro-inflammatory conditions. Its function has been recently described: it codes for immune-responsive gene 1 protein/cis-aconitic acid decarboxylase (IRG1/CAD), an enzyme catalysing the production of itaconic acid from cis-aconitic acid, a tricarboxylic acid (TCA) cycle intermediate. Itaconic acid possesses specific antimicrobial properties inhibiting isocitrate lyase, the first enzyme of the glyoxylate shunt, an anaplerotic pathway that bypasses the TCA cycle and enables bacteria to survive on limited carbon conditions. To elucidate the mechanisms underlying itaconic acid production through IRG1 induction in macrophages, we examined the transcriptional regulation of IRG1. To this end, we studied IRG1 expression in human immune cells under different inflammatory stimuli, such as TNFα and IFNγ, in addition to lipopolysaccharides. Under these conditions, as previously shown in mouse macrophages, IRG1/CAD accumulates in mitochondria. Furthermore, using literature information and transcription factor prediction models, we re-constructed raw gene regulatory networks (GRNs) for IRG1 in mouse and human macrophages. We further implemented a contextualization algorithm that relies on genome-wide gene expression data to infer putative cell type-specific gene regulatory interactions in mouse and human macrophages, which allowed us to predict potential transcriptional regulators of IRG1. Among the computationally identified regulators, siRNA-mediated gene silencing of interferon regulatory factor 1 (IRF1) in macrophages significantly decreased the expression of IRG1/CAD at the gene and protein level, which correlated with a reduced production of itaconic acid. Using a synergistic approach of both computational and experimental methods, we here shed more light on the transcriptional machinery of IRG1 expression and could pave the way to therapeutic approaches targeting itaconic acid levels. [less ▲]

L-plastin Ser5 phosphorylation in breast cancer cells and in vitro is mediated by RSK downstream of the ERK/MAPK pathway

in FASEB Journal (2016)

Deregulated cell migration and invasion are hallmarks of metastatic cancer cells. Phosphorylation on residue Ser5 of the actin-bundling protein L-plastin activates L-plastin and has been reported to be ... [more ▼]

Deregulated cell migration and invasion are hallmarks of metastatic cancer cells. Phosphorylation on residue Ser5 of the actin-bundling protein L-plastin activates L-plastin and has been reported to be crucial for invasion and metastasis. Here, we investigate signal transduction leading to L-plastin Ser5 phosphorylation using 4 human breast cancer cell lines. Whole-genome microarray analysis comparing cell lines with different invasive capacities and corresponding variations in L-plastin Ser5 phosphorylation level revealed that genes of the ERK/MAPK pathway are differentially expressed. It is noteworthy that in vitro kinase assays showed that ERK/MAPK pathway downstream ribosomal protein S6 kinases α-1 (RSK1) and α-3 (RSK2) are able to directly phosphorylate L-plastin on Ser5. Small interfering RNA- or short hairpin RNA-mediated knockdown and activation/inhibition studies followed by immunoblot analysis and computational modeling confirmed that ribosomal S6 kinase (RSK) is an essential activator of L-plastin. Migration and invasion assays showed that RSK knockdown led to a decrease of up to 30% of migration and invasion of MDA-MB-435S cells. Although the presence of L-plastin was not necessary for migration/invasion of these cells, immunofluorescence assays illustrated RSK-dependent recruitment of Ser5-phosphorylated L-plastin to migratory structures. Altogether, we provide evidence that the ERK/MAPK pathway is involved in L-plastin Ser5 phosphorylation in breast cancer cells with RSK1 and RSK2 kinases able to directly phosphorylate L-plastin residue Ser5. [less ▲]

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

in JAK-STAT (2015), 4(1), 1062596

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 ... [more ▼]

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-PDGFRalpha kinase exhibits a significantly different signaling pattern compared to its PDGFRalpha 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-PDGFRalpha restores activation of MAPK- and PI3K-pathways. In stark contrast to the classical cytokine-induced STAT activation process, STAT activation by FIP1L1-PDGFRalpha does neither require Janus kinase activity nor Src kinase activity. Furthermore, we investigated the mechanism of STAT5 activation via FIP1L1-PDGFRalpha 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-PDGFRalpha. [less ▲]

ID: 194: Role of microRNAs in signal transduction pathways of the inflammatory cytokine Interleukin-6: Relevance for liver diseases

in Cytokine (2015), 76(1), 99-

A comprehensive integrative analysis of the transcriptional network underlying the zebrafish heart regeneration

Poster (2014, September 08)

Despite a notable reduction in incidence of acute myocardial infarction (MI), patients who experienced it remain at risk for premature death and cardiac malfunction. The human cardiomyocytes are not able ... [more ▼]

Despite a notable reduction in incidence of acute myocardial infarction (MI), patients who experienced it remain at risk for premature death and cardiac malfunction. The human cardiomyocytes are not able to achieve extensive regeneration upon MI. Remarkably, the adult zebrafish is able to achieve complete heart regeneration following amputation, cryoinjury or genetic ablation. This raises new potential opportunities on how to boost heart healing capacity in humans. The objective of our research is to characterize the transcriptional network of the zebrafish heart regeneration and underlying regulatory mechanisms. To conduct our investigation, we used microarray data from zebrafish at 6 post-cryoinjury time points (4 hours, and 1, 3, 7, 14 and 90 days) and control samples. We thereon looked for the gene co-expression patterns in the data and, based on that, constructed a weighted gene co-expression network. To detect candidate functional sub-networks (modules), we used two different network clustering approaches: a density-based (ClusterONE) and a topological overlap-based (Hybrid Dynamic Branch Cut) algorithms. The visualization of the expression changes of the candidate modules reflected the dynamics of the recovery process. Also we aimed to identify candidate “hub” genes that might regulate the behavior of the biological modules and drive the regeneration process. We identified eighteen distinct modules associated with heart recovery upon cryoinjury. Functional enrichment analysis displayed that the modules are involved in different cellular processes crucial for heart regeneration, including: cell fate specification (p-value < 0.006) and migration (p-value < 0.047), ribosome biogenesis (p-value < 0.004), cardiac cell differentiation (p-value < 3E-04), and various signaling events (p-value < 0.037). The visualization of the modules’ expression profiles confirmed the relevance of these functional enrichments. For instance, the genes of the module involved in regulation of endodermal cell fate specification were up-regulated upon injury until 3 days. Among the candidate hub genes detected in the network, there are genes relevant to atherosclerosis treatment and inflammation during cardiac arrest. These and other findings are currently undergoing deeper computational analyses. The top promising targets will be independently validated using our zebrafish (in vivo) model. In conclusion, our findings provide insights into the complex regulatory mechanisms involved during heart regeneration in the zebrafish. These data will be useful for modelling specific network-based responses to heart injury, and for finding sensitive network points that may trigger or boost heart regeneration. [less ▲]

Glioblastomas are composed of genetically divergent clones with distinct tumourigenic potential and variable stem cell-associated phenotypes.

in Acta Neuropathologica (2014), 127(2), 203-19

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the ... [more ▼]

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36%, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM. [less ▲]

Dynamic regulation of microRNA expression following interferonγ- induced gene transcription

in RNA Biology (2012), 9(7), 987-989

MicroRNAs are major players in post-transcriptional gene regulation. Even small changes in miRNA levels may have profound consequences for the expression levels of target genes. Hence, miRNAs themselves ... [more ▼]

MicroRNAs are major players in post-transcriptional gene regulation. Even small changes in miRNA levels may have profound consequences for the expression levels of target genes. Hence, miRNAs themselves need to be tightly, albeit dynamically, regulated. Here, we investigated the dynamic behavior of miRNAs over a wide time range following stimulation of melanoma cells with interferonγ (IFNγ), which activates the transcription factor STAT1. By applying several bioinformatic and statistical software tools for visualization and identification of differentially expressed miRNAs derived from time-series microarray experiments, 8.9% of 1105 miRNAs appeared to be directly or indirectly regulated by STAT1. Focusing on distinct dynamic expression patterns, we found that the majority of robust miRNA expression changes occurred in the intermediate time range (24-48 h). Three miRNAs (miR-27a, miR-30a and miR-34a) had a delayed regulation occurring at 72 h while none showed significant expression changes at early time points between 30 min and 6 h. Expression patterns of individual miRNAs were altered gradually over time or abruptly increased or decreased between two time points. Furthermore, we observed coordinated dynamic transcription of most miRNA clusters while few were found to be regulated independently of their genetic cluster. Most interestingly, several "star" or passenger strand sequences were specifically regulated over time while their "guide" strands were not. © 2012 Landes Bioscience. [less ▲]

Signatures of MicroRNAs and selected MicroRNA target genes in human melanoma

in Cancer Research (2010), 70(10), 4163-4173

Small noncoding microRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation or orchestrating their sequence-specific degradation. In this study, we investigated miRNA and ... [more ▼]

Small noncoding microRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation or orchestrating their sequence-specific degradation. In this study, we investigated miRNA and miRNA target gene expression patterns in melanoma to identify candidate biomarkers for early and progressive disease. Because data presently available on miRNA expression in melanoma are inconsistent thus far, we applied several different miRNA detection and profiling techniques on a panel of 10 cell lines and 20 patient samples representing nevi and primary or metastatic melanoma. Expression of selected miRNAs was inconsistent when comparing cell line-derived and patient-derived data. Moreover, as expected, some discrepancies were also detected when miRNA microarray data were correlated with qPCR-measured expression levels. Nevertheless, we identified miRNA-200c to be consistently downregulated in melanocytes, melanoma cell lines, and patient samples, whereas miRNA-205 and miRNA-23b were markedly reduced only in patient samples. In contrast, miR-146a and miR-155 were upregulated in all analyzed patients but none of the cell lines. Whole-genome microarrays were performed for analysis of selected melanoma cell lines to identify potential transcriptionally regulated miRNA target genes. Using Ingenuity pathway analysis, we identified a deregulated gene network centered around microphthalmia-associated transcription factor, a transcription factor known to play a key role in melanoma development. Our findings define miRNAs and miRNA target genes that offer candidate biomarkers in human melanoma. ©2010 AACR. [less ▲]