Abstract High-Mobility Group Box 1 (HMGB1) is a chromatin-associated protein critically involved in nucleosome assembly. During cell necrosis, HMGB1 is first translocated from the nucleus to the cytoplasm, and then passively released to the extracellular space, where it initiates and propagates the inflammatory response. Immune cells (e.g. macrophages) and cancer cells are also able to actively secrete HMGB1. Extracellular HMGB1 is therefore considered a crucial molecule for both chronic inflammation and cancer development, and a promising therapeutic target. In this study, using a malignant mesothelioma (MM) model, we tested the hypothesis that the known anti-cancer effects of aspirin (acetyl salicylic acid), the most widely used anti-inflammatory drug, and salicylic acid, its major metabolite, could be due to HMGB1 inhibition. In vitro, we analyzed the effects of salicylates on HMGB1-induced MM cell proliferation, wound healing, migration, invasion and anchorage-independent colony formation. Moreover, using a MM xenograft model, we tested whether in vivo suppression of HMGB1 signaling by salicylates or BoxA, a specific HMGB1 inhibitor, resulted in reduced MM growth and increased survival. Our results demonstrated that salicylates, at doses normally used in clinical practice (≤1 mM), significantly suppress HMGB1-induced in vitro pro-tumorigenic effects on MM cells and inhibits in vivo MM growth, significantly extending mice survival. Our findings are critically relevant to the understanding of the poorly known anti-cancer effects of salicylates. From this perspective, they apply to more than 50 million people in the USA alone currently taking aspirin. Finally, these preclinical results could be translated to improve the current treatment of the usually lethal MM, and of other HMGB1-related malignancies as well. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C187. Citation Format: Andrea Napolitano, Laura Pellegrini, Sandro Jube, Cormac J. Jennings, Erin G. Flores, David Larson, Vishal S. Negi, Ian Pagano, Mika Tanji, Amy Powers, Sandra Pastorino, Harvey I. Pass, Marco E. Bianchi, Michele Carbone, Haining Yang. Salicylates suppress tumor growth via inhibition of HMGB1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C187.
Abstract Malignant Mesothelioma is a highly aggressive cancer, which is difficult to diagnose and treat. Here we describe the molecular, cellular and morphological characterization of a syngeneic system consisting of murine AB1, AB12 and AB22 mesothelioma cells injected in immunocompetent BALB/c mice, which allows the study of the interplay of tumor cells with the immune system. Murine mesothelioma cells, like human ones, respond to exogenous High Mobility Group Box 1 protein, a Damage-Associated Molecular Pattern that acts as a chemoattractant for leukocytes and as a proinflammatory mediator. The tumors derived from AB cells are morphologically and histologically similar to human MM tumors, and respond to treatments used for MM patients. Our system largely recapitulates human mesothelioma, and we advocate its use for the study of MM development and treatment.
The eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-κB) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-κB activity in single cells during infection. Here, we investigated at the single cell level how NF-κB nuclear localization - a proxy for NF-κB activity - oscillates in infected and uninfected fibroblasts co-existing in cultures exposed to Salmonella enterica serovar Typhimurium. Fibroblasts were used due to the capacity of S. Typhimurium to persist in this cell type. Real-time dynamics of NF-κB was examined in microfluidics, which prevents cytokine accumulation. In this condition, infected (ST+) cells translocate NF-κB to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, ST- fibroblasts exhibited higher NF-κB nuclear translocation than the ST+ population, with these latter cells turning refractory to external stimuli such as TNF-α or a second infection. Sorting of ST+ and ST- cell populations confirmed enhanced expression of NF-κB target genes such as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-κB response in the infected fibroblast. Higher expression of SOCS3, encoding a "suppressor of cytokine signaling," was also observed in the ST+ population. Intracellular S. Typhimurium subverts NF-κB activity using protein effectors translocated by the secretion systems encoded by pathogenicity islands 1 (T1) and 2 (T2). T1 is required for regulating expression of SOCS3 and all NF-κB target genes analyzed whereas T2 displayed no role in the control of SOCS3 and IL1B expression. Collectively, these data demonstrate that S. Typhimurium attenuates NF-κB signaling in fibroblasts, an effect only perceptible when ST+ and ST- populations are analyzed separately. This tune-down in a central host defense might be instrumental for S. Typhimurium to establish intracellular persistent infections.
High mobility group B box (HMGB) proteins are a family of chromatin proteins made up of two basic DNA binding domains, HMG box A and B, and a C-terminal acidic tail. HMGB have a highly conserved sequence, but different expression pattern: HMGB1 is almost ubiquitous, whereas the others are highly expressed in only a few tissues in adults. We previously demonstrated that HMGB1 is released by necrotic cells and has chemoattractant activity for inflammatory and stem cells, via binding to receptor for advanced glycation endproducts (RAGE). HMGB1 can be actively secreted by inflammatory cells. Here, we report that also HMGB2 can be secreted by THP-1 cells, and promotes proliferation and migration of endothelial cells. These functions of HMGB2 are exerted via engagement of RAGE, whose blockade completely abrogates cell responses. Since extracellular HMGB2 has been detected in the blood and other biological fluids, it might be necessary to target HMGB2 at the same time as HMGB1 for therapeutical efficacy.
An abnormal generation of reactive oxygen species (ROS) is thought to contribute to systemic sclerosis (SSc), fostering autoimmunity, fibrosis, and vascular inflammation. The function of the prototypic damage-associated molecular pattern, high mobility group box 1 (HMGB1), depends on its redox status. Here we investigate whether oxidative stress regulates the cross-talk between leukocytes and platelets via HMGB1, thus contributing to vessel inflammation in SSc.The oxidation of HMGB1 amplified its ability to activate neutrophils, as detected assessing the redistribution of primary granule molecules and the transactivation of the β2 integrin chain CD18. Activated platelets are a source of bioactive HMGB1 and via P-selectin stimulated neutrophils to generate ROS. Oxidized extracellular HMGB1, soluble or associated to platelet membrane or to platelet-derived microparticles (PDμPs), further increased leukocyte activation. Leukocyte activation abated in the presence of inhibitors of HMGB1 or of catalase, which catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. The redistribution of the content of primary granules and the transactivation of β2 integrins characterized blood leukocytes of SSc patients and membrane HMGB1 was significantly higher in patients with pulmonary hypertension or with diffuse SSc. HMGB1(+) microparticles (μPs) purified from SSc patients, but not HMGB1(-) μPs purified from control subjects, activated in vitro healthy neutrophils, and HMGB1 inhibitors reversed the effects of μPs.ROS dramatically increase the ability of extracellular HMGB1 to activate blood leukocytes. This event might contribute to maintain the microvascular injury of patients with SSc.
Abstract Signals of tissue necrosis, damage-associated molecular patterns (DAMPs), cause inflammation. Leukocytes migrating into injured tissues tonically release DAMPs, including the high mobility group box 1 protein (HMGB1). In the absence of suitable models, the relative role of DAMPs released because of necrosis or leukocyte activation has not, so far, been dissected. We have generated a mouse model lacking Hmgb1 in the hematopoietic system and studied the response to acute sterile injury of the skeletal muscle. Regenerating fibers are significantly less numerous at earlier time points and smaller at the end of the process. Leukocyte Hmgb1 licenses the skeletal muscle to react to hypoxia, to express angiopoietin-2, and to initiate angiogenesis in response to injury. Vascularization of the regenerating tissue is selectively jeopardized in the absence of leukocyte Hmgb1, revealing that it controls the nutrient and oxygen supply to the regenerating tissue. Altogether, our results reveal a novel nonredundant role for leukocyte Hmgb1 in the repair of injured skeletal muscle.
