Aleksandra Ellert-Miklaszewska

Characteristics of the alternative phenotype of microglia/macrophages and its modulation in experimental gliomas.

Microglia (brain resident macrophages) accumulate in malignant gliomas and instead of initiating the anti-tumor response, they switch to a pro-invasive phenotype, support tumor growth, invasion, angiogenesis and immunosuppression by release of cytokines/chemokines and extracellular matrix proteases. Using immunofluorescence and flow cytometry, we demonstrate an early accumulation of activated microglia followed by accumulation of macrophages in experimental murine EGFP-GL261 gliomas. Those cells acquire the alternative phenotype, as evidenced by evaluation of the production of ten pro/anti-inflammatory cytokines and expression profiling of 28 genes in magnetically-sorted CD11b+ cells from tumor tissues. Furthermore, we show that infiltration of implanted gliomas by amoeboid, Iba1-positive cells can be reduced by a systematically injected cyclosporine A (CsA) two or eight days after cell inoculation. The up-regulated levels of IL-10 and GM-CSF, increased expression of genes characteristic for the alternative and pro-invasive phenotype (arg-1, mt1-mmp, cxcl14) in glioma-derived CD11b+ cells as well as enhanced angiogenesis and tumor growth were reduced in CsA-treated mice. Our findings define for the first time kinetics and biochemical characteristics of glioma-infiltrating microglia/macrophages. Inhibition of the alternative activation of tumor-infiltrating macrophages significantly reduced tumor growth. Thus, blockade of microglia/macrophage infiltration and their pro-invasive functions could be a novel therapeutic strategy in malignant gliomas.

MAPK Signal Transduction Underlying Brain Inflammation and Gliosis as Therapeutic Target

A majority, if not all, acute and progressive neurodegenerative diseases are accompanied by local microglia‐mediated inflammation, astrogliosis, infiltration of immune cells, and activation of the adaptive immunity. These processes progress by the expression of cytokines, adhesion molecules, proteases, and other inflammation mediators. In response to brain injury or infection, intracellular signaling pathways are activated in microglia, which turn on inflammatory and antigen‐presenting cell functions. Different extrinsic signals shape microglial activation toward neuroprotective or neurotoxic phenotype under pathological conditions. This review discusses recent advances regarding molecular mechanisms of inflammatory signal transduction in neurological disorders and in in vitro models of inflammation/gliosis. Mitogen‐activated protein kinases (MAPKs) are a family of serine/threonine protein kinases responsible for most cellular responses to cytokines and external stress signals and crucial for regulation of the production of inflammation mediators. Increased activity of MAPKs in activated microglia and astrocytes, and their regulatory role in the synthesis of inflammatory cytokines mediators, make them potential targets for novel therapeutics. MAPK inhibitors emerge as attractive anti‐inflammatory drugs, because they are capable of reducing both the synthesis of inflammation mediators at multiple levels and are effective in blocking inflammatory cytokine signaling. Small molecule inhibitors targeting of p38 MAPK and JNK pathways have been developed and offer a great potential as potent modulators of brain inflammation and gliosis in neurological disorders, where cytokine overproduction contributes to disease progression. Many of the pharmacological MAPK inhibitors can be administered orally and initial results show therapeutic benefits in preclinical animal models. Anat Rec, 292:1902–1913, 2009.

The signal transducers Stat1 and Stat3 and their novel target Jmjd3 drive the expression of inflammatory genes in microglia

Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of pro-inflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-γ signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression.Key MessageCombined analysis of genomic Stat occupancy and transcriptome revealed novel Stat target genes in LPS-induced microglia.Jmjd3 transcription factor is a novel transcriptional target of Stat1 and Stat3.Stat1 and Stat3 cooperate with Jmjd3 to induce the expression of pro-inflammatory genes.Constitutively active Stat1 and Stat3 fully mimic the LPS-induced upregulation of inflammatory genes and secretion of cytokines.

Molecular definition of the pro-tumorigenic phenotype of glioma-activated microglia

Microglia are myeloid cells residing in the central nervous system that participate in inflammatory responses and could promote injury and repair. Gliomas attract microglia and polarize them into tumor‐supporting cells that participate in matrix remodeling, invasion, angiogenesis, and suppression of adaptive immunity. Although signaling pathways and critical regulators underlying classical inflammation are well established, signal transduction and transcriptional circuits underlying the alternative activation of microglia are poorly known. Using primary rat microglial cultures exposed to glioma conditioned medium or lipopolysaccharide (LPS), we demonstrate that microglia adapt different fates and polarize into pro‐inflammatory or alternatively activated cells. Glioma‐derived factors increased cell motility, phagocytosis, and sustained proliferation of microglial cells that was mediated by enhanced focal adhesion kinase and PI‐3K/Akt signaling. The signals from glioma cells induced ERK and p38 MAPK but not JNK signaling and failed to activate pro‐inflammatory Stat1 and NFκB signaling in microglial cells. Transcriptome analysis of microglial cultures at 6 h after exposure to glioma‐conditioned medium or LPS revealed different patterns of gene expression. Glioma‐induced activation was associated with induction of genes coding for ID (inhibitor of DNA binding) 1/3 and c‐Myc, markers of the alternative phenotype Arg1, MT1‐MMP, CXCL14, and numerous cytokines/chemokines implicated in immune cell trafficking. Many classical inflammation‐related genes and signaling pathways failed to be induced. Our study indicates for the first time molecular pathways that direct microglia toward the pro‐invasive, immunosuppressive phenotype.

Non-apoptotic Fas signaling regulates invasiveness of glioma cells and modulates MMP-2 activity via NFκB-TIMP-2 pathway

Fas (CD95/APO-1) is a cell surface death receptor that mediates apoptosis upon engagement by its ligand, FasL. Paradoxically, Fas/FasL can also promote cell invasion among non-apoptotic cells; here, we show that Fas/FasL signaling can promote tumor invasion when apoptosis is compromised. We have developed a recombinant FasL Interfering Protein (FIP) to interfere with Fas signaling in C6 glioma cells expressing both Fas receptor and its ligand. FIP administration did not affect cell viability but impaired cell motility and invasiveness of glioma cells. Blockade of Fas signaling reduced MMP-2 activity in glioma cells, that was associated with down-regulation of MAPK signaling, and AP-1 and NFkappaB-driven transcription. FIP treatment did not affect mmp-2 and mt1-mmp expression but significantly attenuated timp-2 expression and TIMP-2 amount in the culture medium. Studies with pharmacological inhibitors of JNK/c-Jun (SP600125) and NFkappaB (BAY11-7082) signaling pathways demonstrated that timp-2 expression is regulated by NFkappaB transcription factor. Our findings show that non-apoptotic Fas signaling activated in the autocrine manner or through microenvironment derived factors can regulate invasiveness of glioma cells via modulation of MMP-2 activation, likely by controlling TIMP-2 expression.

Cyclosporine a induces growth arrest or programmed cell death of human glioma cells

Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the present study, we found the induction of growth arrest or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies.

Novel small molecular inhibitors disrupt the JAK/STAT3 and FAK signaling pathways and exhibit a potent antitumor activity in glioma cells

JAK (Janus kinase)/STAT (signal transducers and activators of transcription) signaling is involved in the regulation of cell growth, differentiation and apoptosis. Constitutive activation of STATs, in particular STAT3, is observed in a large number of human tumors, including gliomas and may contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis, thus it emerges as a promising target for anti-cancer therapy. To investigate the therapeutic potential of blocking STAT3 in glioma cells a set of small synthetic molecules - caffeic acid derivatives, structurally related to AG490 was screened for its ability to inhibit STAT3. Inhibitor 2 (E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide was the most effective in inhibition of JAK/STAT3 signaling and at doses ≥ 25 μM significantly reduced the level of phosphorylated JAK1, JAK2 and STAT3 (at Tyr705) and downregulated the expression of known STAT3 targets. In treated cells we observed rapid detachment and rounding of cells associated with reduction of focal adhesion kinase phosphorylation and activity, followed by upregulation of phosphorylated p38, JNK and ERK1/2 levels. Accumulation of cells with fragmented DNA, increases of the cleaved caspase 3 and fragmented PARP levels were detected 24 h after the treatment suggesting ongoing apoptotic cell death. Three human malignant glioblastoma cell lines defective in tumor suppressors TP53 and/or PTEN were susceptible to inhibitor 2 that induced the programmed cell death. Global gene expression profiling revealed modulation of numerous genes in cells treated with inhibitor 2 revealing novel, potential JAK/STAT targets. Our study demonstrates that suitably modified caffeic acid molecules exhibit significant cytotoxic potential toward glioma cells.

Computational models of the JAK1/2-STAT1 signaling

Despite a conceptually simple mechanism of signaling, the JAK-STAT pathway exhibits considerable behavioral complexity. Computational pathway models are tools to investigate in detail signaling process. They integrate well with experimental studies, helping to explain molecular dynamics and to state new hypotheses, most often about the structure of interactions. A relatively small amount of experimental data is available for a JAK1/2-STAT1 variant of the pathway, hence, only several computational models were developed. Here we review a dominant approach of kinetic modeling of the JAK1/2-STAT1 pathway, based on ordinary differential equations. We also give a brief overview of attempts to computationally infer topology of this pathway.

Molecular Characterization of STAT Signaling in Inflammation and Tumorigenesis

The Janus kinases (JAK) and signal transducer and activator of transcription (STAT) signaling are strongly activated in many tumors. STAT proteins are activated by phosphorylation at the tyrosine residue, then dimerize, translocate to the nucleus and bind DNA, initiating the transcription of target genes. Activation of JAK-STAT pathway is implicated in the regulation of cell growth, differentiation, survival and cross-talk between cancer and immune cells. The activation of STATs depends on phosphorylation on a single tyrosine residue (e.g., Tyr705 in STAT3 and Tyr694 in STAT5) in the C-terminal domain. Commercially available antibodies discriminate between total and specifically phosphorylated (active) forms of different STATs, which allows to measure directly STATs activation in crude cell extracts. Nuclear translocation and transcriptional activity of STATs can be measured in transfected cells using STAT dependent promoter driving reporter luciferase gene. STAT signaling pathway and STAT-dependent gene expression in cells can be specifically modulated using oligodeoxynucleotide (ODN) STAT decoy which is a double-stranded fragment of DNA containing an overlapping ISRE/GAS binding site.

Short peptides interfering with signaling pathways as new therapeutic tools for cancer treatment

Short peptides have many advantages, such as low molecular weight, selectivity for a specific target, organelles or cells with minimal toxicity. We describe properties of short peptides, which interfere with communication networks in tumor cells and within microenvironment of malignant gliomas, the most common brain tumors. We focus on ligand/receptor axes and intracellular signaling pathways critical for gliomagenesis that could be targeted with interfering peptides. We review structures and efficacy of organelle-specific and cell-penetrating peptides and describe diverse chemical modifications increasing proteolytic stability and protecting synthetic peptides against degradation. We report results of application of short peptides in glioma therapy clinical trials, their rises and falls. The most advanced examples of therapeutics such as short interfering peptides combined with cell-penetrating peptides that show good effectiveness in disease models are presented. It is foreseen that identification of peptides with better clinical properties may improve their success rates in clinical trials.