Jose Basilio

Tissue factor is induced by interleukin-33 in human endothelial cells: a new link between coagulation and inflammation.

Tissue factor (TF) is the primary trigger of coagulation. Elevated levels of TF are found in atherosclerotic plaques, and TF leads to thrombus formation when released upon plaque rupture. Interleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial cells (ECs). Here, we investigated the impact of IL-33 on TF in human ECs, as a possible new link between inflammation and coagulation. IL-33 induced TF mRNA and protein in human umbilical vein ECs and coronary artery ECs. IL-33-induced TF expression was ST2- and NF-κB-dependent, but IL-1-independent. IL-33 also increased cell surface TF activity in ECs and TF activity in ECs-derived microparticles. IL-33-treated ECs reduced coagulation time of whole blood and plasma but not of factor VII-deficient plasma. In human carotid atherosclerotic plaques (n = 57), TF mRNA positively correlated with IL-33 mRNA expression (r = 0.691, p < 0.001). In this tissue, IL-33 and TF protein was detected in ECs and smooth muscle cells by immunofluorescence. Furthermore, IL-33 and TF protein co-localized at the site of clot formation within microvessels in plaques of patients with symptomatic carotid stenosis. Through induction of TF in ECs, IL-33 could enhance their thrombotic capacity and thereby might impact on thrombus formation in the setting of atherosclerosis.

NF-κB contributes to MMP1 expression in breast cancer spheroids causing paracrine PAR1 activation and disintegrations in the lymph endothelial barrier in vitro

RELA, RELB, CREL, NFKB1 and NFKB2, and the upstream regulators NEMO and NIK were knocked-down in lymph endothelial cells (LECs) and in MDA-MB231 breast cancer spheroids to study the contribution of NF-κB in vascular barrier breaching. Suppression of RELA, NFKB1 and NEMO inhibited “circular chemo-repellent induced defects” (CCIDs), which form when cancer cells cross the lymphatic vasculature, by ~20–30%. Suppression of RELB, NFKB2 and NIK inhibited CCIDs by only ~10–15%. In MDA-MB231 cells RELA and NFKB1 constituted MMP1 expression, which caused the activation of PAR1 in adjacent LECs. The knock-down of MMP1 in MDA-MB231 spheroids and pharmacological inhibition of PAR1 in LECs inhibited CCID formation by ~30%. Intracellular Ca2+ release in LECs, which was induced by recombinant MMP1, was suppressed by the PAR1 inhibitor SCH79797, thereby confirming a functional intercellular axis: RELA/NFKB1 – MMP1 (MDA-MB231) – PAR1 (LEC). Recombinant MMP1 induced PAR1-dependent phosphorylation of MLC2 and FAK in LECs, which is indicative for their activity and for directional cell migration such as observed during CCID formation. The combined knock-down of the NF-κB pathways in LECs and MDA-MB231 spheroids inhibited CCIDs significantly stronger than knock-down in either cell type alone. Also the knock-down of ICAM-1 in LECs (a NF-κB endpoint with relevance for CCID formation) and knock-down of MMP1 in MDA-MB231 augmented CCID inhibition. This evidences that in both cell types NF-κB significantly and independently contributes to tumour-mediated breaching of the lymphatic barrier. Hence, inflamed tumour tissue and/or vasculature pose an additional threat to cancer progression.

Sequence-function correlations and dynamics of ERG isoforms. ERG8 is the black sheep of the family.

The transcription factor ERG is known to have divergent roles. On one hand, it acts as differentiation factor of endothelial cells. On the other hand, it has pathological roles in various cancers. Genomic analyses of the ERG gene show that it gives rise to several isoforms. However, functional differences between these isoforms, representing potential reasons for distinct effects in diverse cell types have not been addressed in detail so far. We set out to investigate the major protein isoforms and found that ERG8 contains a unique C-terminus. This isoform, when expressed as GFP-fusion protein, localized mainly to the cytosol, whereas the other major isoforms (ERG1-4) were predominantly nuclear. Using site directed mutagenesis and laser scanning microscopy of live cells, we could identify nuclear localization (NLS) and nuclear export sequences (NES). These analyses indicated that ERG8 lacks a classical NLS and the DNA-binding domain, but holds an additional NES within its distinctive C-terminus. All the tested isoforms were shuttling between nucleus and cytosol and showed a high degree of mobility. ERG’s 1 to 4 were transcriptionally active on ERG-promoter elements whereas ERG8 was inactive, which is in line with the absence of a DNA-binding domain. Fluorescence resonance energy transfer (FRET) microscopy revealed that ERG8 can bind to the transcriptionally active ERG’s. Knockdown of ERG8 in endothelial cells resulted in upregulation of endogenous ERG-transcriptional activity implying ERG8 as an inhibitor of the active ERG isoforms. Quantitative PCR revealed a different ratio of active ERG’s to ERG8 in cancer- versus non-transformed cells.

Maintenance therapy with histamine plus IL-2 induces a striking expansion of two CD56 bright NK cell subpopulations in patients with acute myeloid leukemia and supports their activation

Histamine dihydrochloride (HDC) plus IL-2 has been proposed as a novel maintenance-immunotherapy in acute myeloid leukemia (AML). We analyzed the immunophenotype and function of natural killer (NK) cells in blood of AML patients treated after chemotherapy with HDC plus IL-2. The treatment caused a striking expansion of CD56brightCD16neg and CD56brightCD16low NK cell subpopulations. A reduced NK cell fraction recovered and high proportions of cells expressed the activating receptors NKG2D, NKp30, and NKp46. Concomitantly, KIR-expressing NK cells were reduced and NK cells with inhibitory NKG2A/CD94 receptors increased beyond normal levels. In addition, the immunotherapy-induced NK cells exhibited high capacity to produce IFN-γ and to degranulate. Furthermore, we provide evidence from subsequent in vitro studies that this is caused in part by direct effects of IL-2 on the CD56bright cells. IL-2 specifically induced proliferation of both CD56bright subpopulations, but not of CD56dim cells. It further preserved the expression of activating receptors and the capacity to produce IFN-γ and to degranulate. These data suggest that therapy with HDC plus IL-2 supports the reconstitution of a deficient NK cell fraction through the specific amplification of CD56bright NK cells giving rise to a functional NK cell compartment with high potential to combat leukemic disease.

TNFα-induced down-regulation of Sox18 in endothelial cells is dependent on NF-κB.

The transcription factor Sox18 plays a role in angiogenesis, including lymphangiogenesis, where it is upregulated by growth factors and directs the expression of genes encoding, e.g., guidance molecules and a matrix metalloproteinase. Conversely, we found that in human umbilical vein endothelial cells (HUVEC) Sox18 is repressed by the pro-inflammatory mediator TNFα (as well as IL-1 and LPS). Since a common feature of these mediators is the activation of the NF-κB signaling pathway, we investigated whether Sox18 downregulation is dependent on this transcription factor. Transduction of HUVEC with an adenoviral vector directing the expression of the NF-κB inhibitor IκBα prevented the downregulation of Sox18. Transient transfections of Sox18 promoter reporter genes revealed that the downregulation takes place on the level of transcription, and that the p65/RelA subunit of NF-κB was operative. Furthermore, the responsible promoter region of Sox18 is located within -1.0kb from the transcriptional start site. The repression of Sox18 and its target genes may lead to altered formation of vessels in inflamed settings.

Myeloid PTEN deficiency impairs tumor-immune surveillance via immune-checkpoint inhibition.

ABSTRACT Tumor–host interaction is determined by constant immune surveillance, characterized by tumor infiltration of myeloid and lymphoid cells. A malfunctioning or diverted immune response promotes tumor growth and metastasis. Recent advances had been made, by treating of certain tumor types, such as melanoma, with T-cell checkpoint inhibitors. This highlights the importance of understanding the molecular mechanisms underlying the crosstalk between tumors and their environment, in particular myeloid and lymphoid cells. Our aim was to study the contribution of the myeloid PI3K/PTEN-signaling pathway in the regulation of tumor-immune surveillance in murine models of cancer. We made use of conditional PTEN-deficient mice, which exhibit sustained activation of the PI3K-signaling axis in a variety of myeloid cell subsets such as macrophages and dendritic cells (DCs). In colitis-associated colon cancer (CAC), mice deficient in myeloid PTEN showed a markedly higher tumor burden and decreased survival. We attributed this observation to the increased presence of immune-modulatory conventional CD8α+ DCs in the spleen, whereas other relevant myeloid cell subsets were largely unaffected. Notably, we detected enhanced surface expression of PD-L1 and PD-L2 on these DCs. As a consequence, tumoricidal T-cell responses were hampered or redirected. Taken together, our findings indicated an unanticipated role for the PI3K/PTEN-signaling axis in the functional regulation of splenic antigen-presenting cells (APCs). Our data pointed at potential, indirect, tumoricidal effects of subclass-specific PI3K inhibitors, which are currently under clinical investigation for treatment of tumors, via myeloid cell activation.

Androgen receptor dampens tissue factor expression via nuclear factor-κB and early growth response protein 1

Background Prostate cancer is one of the leading causes of cancer death in men. Advanced prostate cancer is usually treated by androgen deprivation therapy (ADT), which aims at diminishing circulating testosterone to reduce cancer growth. There is growing evidence that ADT can increase the rate of venous thromboembolism (VTE) in prostate cancer patients. The tissue factor (TF) gene is one of the A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. most important mediators of coagulation and VTE, but so far there is limited data on androgen receptor (AR) mediated TF gene expression.Essentials Androgen deprivation increases the rate of venous thromboembolism in prostate cancer patients. We characterized androgen receptor‐mediated tissue factor regulation in prostate epithelial cells. Androgen receptor is dampening tissue factor expression in prostate epithelial cells. Androgen deprivation could enhance tissue factor expression and raise venous thromboembolism rates.

Abstract 527: Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition

In the current study we are investigating the effects of PTEN-deficient myeloid cells on tumor immune surveillance. We could previously show that hyper-activation of the PI3K signaling cascade by genetic knock-out of the counteracting phosphatase PTEN induced an anti-inflammatory phenotype in myeloid cells. This resulted in protection of conditional knock-out mice in models of acute infection and inflammation. A reduction in pro-inflammatory responses could however increase tumor burden. To address this question we induced colitis associated colon cancer in conditional PTEN-KO mice and found an increase in tumor burden and a reduction in survival in male KO mice. This was accompanied by increased numbers of splenic antigen-presenting cells (APC) expressing the immune checkpoint regulators PD-L1 and PD-L2. Furthermore, transcriptome analysis in these cells revealed a shift towards gene expression profiles found in professional APCs capable of cross-presentation. As expected, ex-vivo stimulated T-cells from KO-mice showed a reduction in proliferative capacity. These findings were further substantiated by findings in a second tumor model using implanted B16 melanoma cells. In this model myeloid PTEN-deficient mice showed a decrease in T-cell activation and a reduction in melanoma cell killing capacity. Taken together, our findings show that genetic deletion of PTEN in cells of myeloid origin increases splenic APCs expressing immune checkpoint regulators resulting in a decrease in tumor immune surveillance. Our study shows that PI3K-inhibitors which are currently tested as anti-cancer drugs might have additional beneficial effects on immune cells by shifting their inflammatory phenotype. Citation Format: Mario Kuttke, Emine Sahin, Julia Pisoni, Sophie Percig, Andrea Vogel, Daniel Kraemmer, Leslie Hanzl, Julia Stefanie Brunner, Hannah Paar, Klara Soukup, Angela Halfmann, Alexander Dohnal, Carl-Walter Steiner, Stephan Bluml, Jose Basilio, Bernhard Hochreiter, Manuel Salzmann, Bastian Hoesel, Gunther Lametschwandtner, Robert Eferl, Johannes Schmid, Gernot Schabbauer. Myeloid PTEN deficiency impairs tumor immune surveillance via immune checkpoint inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 527.