Proteometabolomics of initial and recurrent glioblastoma highlights an increased immune cell signature with altered lipid metabolism.

BACKGROUND: There is an urgent need to better understand the mechanisms associated with the development, progression, and onset of recurrence after initial surgery in glioblastoma (GBM). The use of integrative phenotype-focused -omics technologies such as proteomics and lipidomics provides an unbiased approach to explore the molecular evolution of the tumor and its associated environment. METHODS: We assembled a cohort of patient-matched initial (iGBM) and recurrent (rGBM) specimens of resected GBM. Proteome and metabolome composition were determined by mass spectrometry-based techniques. We performed neutrophil-GBM cell coculture experiments to evaluate the behavior of rGBM-enriched proteins in the tumor microenvironment. ELISA-based quantitation of candidate proteins was performed to test the association of their plasma concentrations in iGBM with the onset of recurrence. RESULTS: Proteomic profiles reflect increased immune cell infiltration and extracellular matrix reorganization in rGBM. ASAH1, SYMN, and GPNMB were highly enriched proteins in rGBM. Lipidomics indicates the downregulation of ceramides in rGBM. Cell analyses suggest a role for ASAH1 in neutrophils and its localization in extracellular traps. Plasma concentrations of ASAH1 and SYNM show an association with time to recurrence. CONCLUSIONS: We describe the potential importance of ASAH1 in tumor progression and development of rGBM via metabolic rearrangement and showcase the feedback from the tumor microenvironment to plasma proteome profiles. We report the potential of ASAH1 and SYNM as plasma markers of rGBM progression. The published datasets can be considered as a resource for further functional and biomarker studies involving additional -omics technologies.

A jack of all trades - ADAM8 as a signaling hub in inflammation and cancer.

As a member of the family of A Disintegrin And Metalloproteinases (ADAM) ADAM8 is preferentially expressed in lymphatic organs, immune cells, and tumor cells. The substrate spectrum for ADAM8 proteolytic activity is not exclusive but is related to effectors of inflammation and signaling in the tumor microenvironment. In addition, complexes of ADAM8 with extracellular binding partners such as integrin ß-1 cause an extensive intracellular signaling in tumor cells, thereby activating kinase pathways with STAT3, ERK1/2, and Akt signaling, which causes increased cell survival and enhanced motility. The cytoplasmic domain of ADAM8 harbors five SRC homology-3 (SH3) domains that can potentially interact with several proteins involved in actin dynamics and cell motility, including Myosin 1F (MYO1F), which is essential for neutrophil motility. The concept of ADAM8 thus involves immune cell recruitment, in most cases leading to an enhancement of inflammatory (asthma, COPD) and tumor (including pancreatic and breast cancers) pathologies. In this review, we report on available studies that qualify ADAM8 as a therapeutic target in different pathologies. As a signaling hub, ADAM8 controls extracellular, intracellular, and intercellular communication, the latter one mainly mediated by the release of extracellular vesicles with ADAM8 as cargo. Here, we will dissect the contribution of different domains to these distinct ways of communication in several pathologies. We conclude that therapeutic targeting attempts for ADAM8 should consider blocking more than a single domain and that this requires a thorough evaluation of potent molecules targeting ADAM8 in an in vivo setting.

ADAM8 signaling drives neutrophil migration and ARDS severity.

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8-/- mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

ADAM8-Dependent Extracellular Signaling in the Tumor Microenvironment Involves Regulated Release of Lipocalin 2 and MMP-9.

The metalloprotease-disintegrin ADAM8 is critically involved in the progression of pancreatic cancer. Under malignant conditions, ADAM8 is highly expressed and could play an important role in cell-cell communication as expression has been observed in tumor and immune cells of the tumor microenvironment (TME) such as macrophages. To analyze the potential role of ADAM8 in the TME, ADAM8 knockout PDAC tumor cells were generated, and their release of extracellular vesicles (EVs) was analyzed. In EVs, ADAM8 is present as an active protease and associated with lipocalin 2 (LCN2) and matrix metalloprotease 9 (MMP-9) in an ADAM8-dependent manner, as ADAM8 KO cells show a lower abundance of LCN2 and MMP-9. Sorting of ADAM8 occurs independent of TSG101, even though ADAM8 contains the recognition motif PTAP for the ESCRTI protein TSG101 within the cytoplasmic domain (CD). When tumor cells were co-cultured with macrophages (THP-1 cells), expression of LCN2 and MMP-9 in ADAM8 KO cells was induced, suggesting that macrophage signaling can overcome ADAM8-dependent intracellular signaling in PDAC cells. In co-culture with macrophages, regulation of MMP-9 is independent of the M1/M2 polarization state, whereas LCN2 expression is preferentially affected by M1-like macrophages. From these data, we conclude that ADAM8 has a systemic effect in the tumor microenvironment, and its expression in distinct cell types has to be considered for ADAM8 targeting in tumors.

Extracellular Vesicle-Based Detection of Pancreatic Cancer.

Due to a grim prognosis, there is an urgent need to detect pancreatic ductal adenocarcinoma (PDAC) prior to metastasis. However, reliable diagnostic imaging methods or biomarkers for PDAC or its precursor lesions are still scarce. ADAM8, a metalloprotease-disintegrin, is highly expressed in PDAC tissue and negatively correlates with patient survival. The aim of our study was to determine the ability of ADAM8-positive extracellular vesicles (EVs) and cargo microRNAs (miRNAs) to discriminate precursor lesions or PDAC from healthy controls. In order to investigate enrichment of ADAM8 on EVs, these were isolated from serum of patients with PDAC (n = 52), precursor lesions (n = 7) and healthy individuals (n = 20). Nanoparticle Tracking Analysis and electron microscopy indicated successful preparation of EVs that were analyzed for ADAM8 by FACS. Additionally, EV cargo analyses of miRNAs from the same serum samples revealed the presence of miR-720 and miR-451 by qPCR and was validated in 20 additional PDAC samples. Statistical analyses included Wilcoxon rank test and ROC curves. FACS analysis detected significant enrichment of ADAM8 in EVs from patients with PDAC or precursor lesions compared to healthy individuals (p = 0.0005). ADAM8-dependent co-variates, miR-451 and miR-720 were also diagnostic, as patients with PDAC had significantly higher serum levels of miR-451 and lower serum levels of miR-720 than healthy controls and reached high sensitivity and specificity (AUC = 0.93 and 1.00, respectively) to discriminate PDAC from healthy control. Thus, detection of ADAM8-positive EVs and related cargo miR-720 and miR-451 may constitute a specific biomarker set for screening individuals at risk for PDAC.

Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro.

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1ß, TNF-α, IFN-γ, and TGF-ß. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators.

Cohort Analysis of ADAM8 Expression in the PDAC Tumor Stroma.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer type with one of the highest mortalities. The metalloprotease-disintegrin ADAM8 is highly expressed in pancreatic cancer cells and is correlated with an unfavorable patient prognosis. However, no information is available on ADAM8 expression in cells of the tumor microenvironment. We used immunohistochemistry (IHC) to describe the stromal cell types expressing ADAM8 in PDAC patients using a cohort of 72 PDAC patients. We found ADAM8 expressed significantly in macrophages (6%), natural killer cells (40%), and neutrophils (63%), which showed the highest percentage of ADAM8 expressing stromal cells. We quantified the amount of ADAM8+ neutrophils in post-capillary venules in PDAC sections by IHC. Notably, the amount of ADAM8+ neutrophils could be correlated with post-operative patient survival times. In contrast, neither the total neutrophil count in peripheral blood nor the neutrophil-to-lymphocyte ratio showed a comparable correlation. We conclude from our data that ADAM8 is, in addition to high expression levels in tumor cells, present in tumor-associated stromal macrophages, NK cells, and neutrophils and, in addition to functional implications, the ADAM8-expressing neutrophil density in post-capillary venules is a diagnostic parameter for PDAC patients when the numbers of ADAM8+ neutrophils are quantified.

Discovery of Dimeric Arylsulfonamides as Potent ADAM8 Inhibitors.

The metalloproteinase ADAM8 is upregulated in several cancers but has a dispensable function under physiological conditions. In tumor cells, ADAM8 is involved in invasion, migration, and angiogenesis. The use of bivalent inhibitors could impair migration and invasion through the double binding to a homodimeric form of ADAM8 located on the cell surface of tumor cells. Herein we report the rational design and synthesis of the first dimeric ADAM8 inhibitors selective over ADAM10 and matrix metalloproteinases. Bivalent derivatives have been obtained by dimerizing the structure of a previously described ADAM17 inhibitor, JG26. In particular, derivative 2 was shown to inhibit ADAM8 proteolytic activity in vitro and in cell-based assays at nanomolar concentration. Moreover, it was more effective than the parent monomeric compound in blocking invasiveness in the breast cancer MDA-MB-231 cell line, thus supporting our hypothesis about the importance of inhibiting the active homodimer of ADAM8.

The Relationship between CrossFit® Performance and Laboratory-Based Measurements of Fitness.

To date, research has examined the physiological determinants of performance in standardized CrossFit® (CF) workouts but not without the influence of CF familiarity. Therefore, the purpose of this present study was to examine the predictive value of aerobic fitness, body composition, and total body strength on performance of two standardized CF workouts in CF-naïve participants. Twenty-two recreationally trained individuals (males = 13, females = 9) underwent assessments of peak oxygen consumption (VO2 peak), ventilatory thresholds, body composition, and one repetition maximum tests for the back squat, deadlift, and overhead press in which the sum equaled the CF Total. Participants also performed two CF workouts: a scaled version of the CF Open workout 19.1 and a modified version of the CF Benchmark workout Fran to determine scores based on total repetitions completed and time-to-completion, respectively. Simple Pearson's r correlations were used to determine the relationships between CF performance variables (19.1 and modified Fran) and the independent variables. A forward stepwise multiple linear regression analysis was performed and significant variables that survived the regression analysis were used to create a predictive model of CF performance. Absolute VO2 peak was a significant predictor of 19.1 performance, explaining 39% of its variance (adjusted R2 = 0.39, p = 0.002). For modified Fran, CF Total was a significant predictor and explained 33% of the variance in performance (adjusted R2 = 0.33, p = 0.005). These results suggest, without any influence of CF familiarity or experience, that performance in these two CF workouts could be predicted by distinct laboratory-based measurements of fitness.

ADAM8 in invasive cancers: links to tumor progression, metastasis, and chemoresistance.

Ectodomain shedding of extracellular and membrane proteins is of fundamental importance for cell-cell communication in neoplasias. A Disintegrin And Metalloproteinase (ADAM) proteases constitute a family of multifunctional, membrane-bound proteins with traditional sheddase functions. Their protumorigenic potential has been attributed to both, essential (ADAM10 and ADAM17) and 'dispensable' ADAM proteases (ADAM8, 9, 12, 15, and 19). Of specific interest in this review is the ADAM proteinase ADAM8 that has been identified as a significant player in aggressive malignancies including breast, pancreatic, and brain cancer. High expression levels of ADAM8 are associated with invasiveness and predict a poor patient outcome, indicating a prognostic and diagnostic potential of ADAM8. Current knowledge of substrates and interaction partners gave rise to the hypothesis that ADAM8 dysregulation affects diverse processes in tumor biology, attributable to different functional cores of the multidomain enzyme. Proteolytic degradation of extracellular matrix (ECM) components, cleavage of cell surface proteins, and subsequent release of soluble ectodomains promote cancer progression via induction of angiogenesis and metastasis. Moreover, there is increasing evidence for significance of a non-proteolytic function of ADAM8. With the disintegrin (DIS) domain ADAM8 binds integrins such as ß1 integrin, thereby activating integrin signaling pathways. The cytoplasmic domain is critical for that activation and involves focal adhesion kinase (FAK), extracellular regulated kinase (ERK1/2), and protein kinase B (AKT/PKB) signaling, further contributing to cancer progression and mediating chemoresistance against first-line therapies. This review highlights the remarkable effects of ADAM8 in tumor biology, concluding that pharmacological inhibition of ADAM8 represents a promising therapeutic approach not only for monotherapy, but also for combinatorial therapies.