N-Glycosylation as a Tool to Study Antithrombin Secretion, Conformation, and Function.

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.

Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults.

Exosomes are extracellular vesicles that contain nucleic acids, lipids and metabolites, and play a critical role in health and disease as mediators of intercellular communication. The majority of extracellular vesicles in the blood are platelet-derived. Compared to adults, neonatal platelets are hyporeactive and show impaired granule release, associated with defects in Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Since these proteins participate in biogenesis of exosomes, we investigated the potential differences between newborn and adult plasma-derived exosomes. Plasma-derived exosomes were isolated by ultracentrifugation of umbilical cord blood from full-term neonates or peripheral blood from adults. Exosome characterization included size determination by transmission electron microscopy and quantitative proteomic analysis. Plasma-derived exosomes from neonates were significantly smaller and contained 65% less protein than those from adults. Remarkably, 131 proteins were found to be differentially expressed, 83 overexpressed and 48 underexpressed in neonatal (vs. adult) exosomes. Whereas the upregulated proteins in plasma exosomes from neonates are associated with platelet activation, coagulation and granule secretion, most of the underexpressed proteins are immunoglobulins. This is the first study showing that exosome size and content change with age. Our findings may contribute to elucidating the potential "developmental hemostatic mismatch risk" associated with transfusions containing plasma exosomes from adults.

Transcriptomic rationale for synthetic lethality-targeting ERCC1 and CDKN1A in chronic myelomonocytic leukaemia.

Despite the absence of mutations in the DNA repair machinery in myeloid malignancies, the advent of high-throughput sequencing and discovery of splicing and epigenetics defects in chronic myelomonocytic leukaemia (CMML) prompted us to revisit a pathogenic role for genes involved in DNA damage response. We screened for misregulated DNA repair genes by enhanced RNA-sequencing on bone marrow from a discovery cohort of 27 CMML patients and 9 controls. We validated 4 differentially expressed candidates in CMML CD34+ bone marrow selected cells and in an independent cohort of 74 CMML patients, mutationally contextualized by targeted sequencing, and assessed their transcriptional behavior in 70 myelodysplastic syndrome, 66 acute myeloid leukaemia and 25 chronic myeloid leukaemia cases. We found BAP1 and PARP1 down-regulation to be specific to CMML compared with other related disorders. Chromatin-regulator mutated cases showed decreased BAP1 dosage. We validated a significant over-expression of the double strand break-fidelity genes CDKN1A and ERCC1, independent of promoter methylation and associated with chemorefractoriness. In addition, patients bearing mutations in the splicing component SRSF2 displayed numerous aberrant splicing events in DNA repair genes, with a quantitative predominance in the single strand break pathway. Our results highlight potential targets in this disease, which currently has few therapeutic options.

Circulating small-sized endothelial microparticles as predictors of clinical outcome after chemotherapy for breast cancer: an exploratory analysis.

PURPOSE: Therapeutic exploitation of angiogenesis in breast cancer has been limited by the lack of reliable biomarkers. Circulating small-sized endothelial microparticles (sEMP) are likely to play a significant role as messengers of angiogenesis. Higher levels of EMP have been observed in cancer patients, but their prognostic value in breast cancer is unknown. Our aim was to determine the value of circulating sEMP as a marker of response to chemotherapy in breast cancer. METHODS: We included patients with breast cancer treated with neoadjuvant or first-line chemotherapy. Baseline and post-treatment circulating sEMP (CD144+) were quantified using a flow cytometer approach specifically designed for analysis of small-sized particles (0.1-0.5 µm). Small-sized EMP response was defined as a post-treatment decrease of sEMP larger than the median decrease of sEMP after chemotherapy. Baseline and post-chemotherapy VEGFA levels were determined with ELISA. RESULTS: Forty-four breast cancer patients were included (19 with metastatic and 25 with locally advanced disease). Median levels of sEMP decreased after chemotherapy (P = 0.005). Response to chemotherapy showed a non-significant trend to associate with sEMP response (P = 0.056). A sEMP response was observed in 51% of patients and was associated with better overall survival (HR 0.18; 95% CI 0.04-0.87; P = 0.02) and progression free survival (HR 0.30; 95% CI 0.09-0.99; P = 0.04) in the group of women with metastatic disease. Post-chemotherapy decrease of VEGFA levels was not associated with breast cancer prognosis. CONCLUSIONS: Our results did not support sEMP as a marker of response to chemotherapy. However, our exploratory analysis suggests that in patients with metastatic breast cancer, the decrease of sEMP levels after chemotherapy is associated with better overall and disease free survival and might be superior to VEGFA levels as an angiogenesis-related prognostic marker.

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1.

Proteins containing C2 domains are the sensors for Ca(2+) and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca(2+) binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands ß3-ß4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca(2+)-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.

Effects of conventional neoadjuvant chemotherapy for breast cancer on tumor angiogenesis.

The effects of breast cancer conventional chemotherapy on tumor angiogenesis need to be further characterized. Neoadjuvant chemotherapy is an ideal model to evaluate the results of chemotherapy, allowing intra-patient direct comparison of antitumor and antiangiogenic effects. We sought to analyze the effect of neoadjuvant chemotherapy on tumor angiogenesis and its clinical significance in breast cancer. Breast cancer patients (n = 108) treated with neoadjuvant sequential anthracyclines and taxanes were studied. Pre- and post-chemotherapy microvessel density (MVD) and mean vessel size (MVS) were analyzed after CD34 immunohistochemistry and correlated with tumor expression of pro- and antiangiogenic factors (VEGFA, THBS1, HIF1A, CTGF, and PDGFA) by qRT-PCR. Angiogenic measures at diagnosis varied among breast cancer subtypes. Pre-treatment higher MVS was associated with triple-negative subtype and more advanced disease. Higher MVS was correlated with higher VEGFA (p = 0.003), while higher MVD was correlated with lower antiangiogenic factors expression (THBS1, p < 0.0001; CTGF, p = 0.001). Increased angiogenesis at diagnosis (high MVS and glomeruloid microvascular proliferation) and higher VEGFA expression were associated with tumor recurrence (p = 0.048 and 0.009, respectively). Chemotherapy-induced angiogenic response (defined as decreased MVD) was present in 35.2 % of patients. This response correlated with an increase in antiangiogenic factors (THBS1) without changes in VEGFA expression, and it was associated with tumor downstaging, but not with clinical response, pathologic complete response, or prognosis. Global effects of chemotherapy mainly consisted in an increased expression of antiangiogenic factors (THBS1, CTGF), with significant changes neither of tumor VEGFA nor of MVS. Conventionally scheduled neoadjuvant chemotherapy exerts antiangiogenic effects, through an increase in antiangiogenic factors, THBS1 and CTGF, but the expression of VEGFA is maintained after treatment. Better markers of angiogenic response and a better understanding of the cooperation of chemotherapy and antiangiogenic therapy in the neoadjuvant clinical scenario are needed.

Monastrol suppresses invasion and metastasis in human colorectal cancer cells by targeting fascin independent of kinesin-Eg5 pathway.

Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.

Association of anthracycline-related cardiac histological lesions with NADPH oxidase functional polymorphisms.

OBJECTIVE: Treatment with anthracyclines may cause cardiac dysfunction, but the sequence of anthracycline-induced heart lesions has been incompletely characterized. NADPH oxidase, a key mediator of oxidative cardiac damage and remodeling, modulates anthracycline clinical cardiotoxicity. Our aim was to determine which cardiac histological lesions are specifically induced by anthracycline treatment and to investigate the role of NADPH functional genetic polymorphisms in their development. PATIENTS AND METHODS: Using a retrospective case-control design, we evaluated cardiac histological lesions and NADPH genotype (polymorphisms rs1883112, rs4673, and rs13058338) in 97 consecutive decedents with a cancer diagnosis (48 treated with anthracyclines). RESULTS: Myocytolysis (60%), patched myocardial necrosis (19%), and myocardial fibrosis (diffuse and patched; 62% and 23%, respectively) were associated with anthracycline treatment. In patients receiving anthracyclines, NADPH oxidase polymorphism rs4673 protected against focal myocardial necrosis (odds ratio [OR], 0.11; 95% confidence interval [CI], 0.20-0.63) whereas rs1883112 was strongly associated with cardiac fibrosis (OR, 5.11; 95% CI, 1.59-16.43), which was present in all homozygotes. CONCLUSION: Anthracyclines induce a cardiac remodeling pattern characterized by interstitial or patched fibrosis. The contribution of the functionally relevant NADPH polymorphisms rs1883112 and rs4673 to anthracycline-related heart lesions provides a plausible explanation for their modulation of cardiotoxicity. If confirmed, these findings may lead to better individualized strategies for early detection and prevention of anthracycline cardiotoxicity.

A new prognostic model including immune biomarkers, genomic proliferation tumor markers (AURKA and MYBL2) and clinical-pathological features optimizes prognosis in neoadjuvant breast cancer patients.

Background: Up to 30% of breast cancer (BC) patients treated with neoadjuvant chemotherapy (NCT) will relapse. Our objective was to analyze the predictive capacity of several markers associated with immune response and cell proliferation combined with clinical parameters. Methods: This was a single-center, retrospective cohort study of BC patients treated with NCT (2001-2010), in whom pretreatment biomarkers were analyzed: neutrophil-to-lymphocyte ratio (NLR) in peripheral blood, CD3+ tumor-infiltrating lymphocytes (TILs), and gene expression of AURKA, MYBL2 and MKI67 using qRT-PCR. Results: A total of 121 patients were included. Median followup was 12 years. In a univariate analysis, NLR, TILs, AURKA, and MYBL2 showed prognostic value for overall survival. In multivariate analyses, including hormone receptor, HER2 status, and response to NCT, NLR (HR 1.23, 95% CI 1.01-1.75), TILs (HR 0.84, 95% CI 0.73-0.93), AURKA (HR 1.05, 95% CI 1.00-1.11) and MYBL2 (HR 1.19, 95% CI 1.05-1.35) remained as independent predictor variables. Conclusion: Consecutive addition of these biomarkers to a regression model progressively increased its discriminatory capacity for survival. Should independent cohort studies validate these findings, management of early BC patients may well be changed.

Venetoclax is a potent hepsin inhibitor that reduces the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells.

Introduction: Hepsin is a type II transmembrane serine protease and its expression has been linked to greater tumorigenicity and worse prognosis in different tumors. Recently, our group demonstrated that high hepsin levels from primary tumor were associated with a higher risk of metastasis and thrombosis in localized colorectal cancer patients. This study aims to explore the molecular role of hepsin in colorectal cancer. Methods: Hepsin levels in plasma from resected and metastatic colorectal cancer patients were analyzed by ELISA. The effect of hepsin levels on cell migration, invasion, and proliferation, as well as on the activation of crucial cancer signaling pathways, was performed in vitro using colorectal cancer cells. A thrombin generation assay determined the procoagulant function of hepsin from these cells. A virtual screening of a database containing more than 2000 FDA-approved compounds was performed to screen hepsin inhibitors, and selected compounds were tested in vitro for their ability to suppress hepsin effects in colorectal cancer cells. Xenotransplantation assays were done in zebrafish larvae to study the impact of venetoclax on invasion promoted by hepsin. Results: Our results showed higher plasma hepsin levels in metastatic patients, among which, hepsin was higher in those suffering thrombosis. Hepsin overexpression increased colorectal cancer cell invasion, Erk1/2 and STAT3 phosphorylation, and thrombin generation in plasma. In addition, we identified venetoclax as a potent hepsin inhibitor that reduced the metastatic and prothrombotic phenotypes of hepsin-expressing colorectal cancer cells. Interestingly, pretreatment with Venetoclax of cells overexpressing hepsin reduced their invasiveness in vivo. Discussion: Our results demonstrate that hepsin overexpression correlates with a more aggressive and prothrombotic tumor phenotype. Likewise, they demonstrate the antitumor role of venetoclax as a hepsin inhibitor, laying the groundwork for molecular-targeted therapy for colorectal cancer.

Potential Utility of Induced Translocation of Engineered Bacteria as a Therapeutic Agent for Mounting a Personalized Neoantigen-Based Tumor Immune Response.

Today, an unprecedented understanding of the cancer genome, along with major breakthroughs in oncoimmunotherapy, and a resurgence of nucleic acid vaccines against cancer are being achieved. However, in most cases, the immune system response is still insufficient to react against cancer, especially in those tumors showing low mutational burden. One way to counteract tumor escape can be the induction of bacterial translocation, a phenomenon associated with autoimmune diseases which consists of a leakage in the colonic mucosa barrier, causing the access of gut bacteria to sterile body compartments such as blood. Certain commensal or live-attenuated bacteria can be engineered in such a way as to contain nucleic acids coding for tumor neoantigens previously selected from individual tumor RNAseq data. Hypothetically, these modified bacteria, previously administered orally to a cancer patient, can be translocated by several compounds acting on colonic mucosa, thus releasing neoantigens in a systemic environment in the context of an acute inflammation. Several strategies for selecting neoantigens, suitable bacteria strains, genetic constructs, and translocation inducers to achieve tumor-specific activations of CD4 and CD8 T-cells are discussed in this hypothesis.

Design of Personalized Neoantigen RNA Vaccines Against Cancer Based on Next-Generation Sequencing Data.

The good clinical results of immune checkpoint inhibitors (ICIs) in recent cancer therapy and the success of RNA vaccines against SARS-nCoV2 have provided important lessons to the scientific community. On the one hand, the efficacy of ICI depends on the number and immunogenicity of tumor neoantigens (TNAs) which unfortunately are not abundantly expressed in many cancer subtypes. On the other hand, novel RNA vaccines have significantly improved both the stability and immunogenicity of mRNA and its efficient delivery, this way overcoming past technique limitations and also allowing a quick vaccine development at the same time. These two facts together have triggered a resurgence of therapeutic cancer vaccines which can be designed to include individual TNAs and be synthesized in a timeframe short enough to be suitable for the tailored treatment of a given cancer patient.In this chapter, we explain the pipeline for the synthesis of TNA-carrying RNA vaccines which encompasses several steps such as individual tumor next-generation sequencing (NGS), selection of immunogenic TNAs, nucleic acid synthesis, drug delivery systems, and immunogenicity assessment, all of each step comprising different alternatives and variations which will be discussed.

Antitumoral Effects of Tricyclic Antidepressants: Beyond Neuropathic Pain Treatment.

Growing evidence shows that nerves play an active role in cancer development and progression by altering crucial molecular pathways and cell functions. Conversely, the use of neurotropic drugs, such as tricyclic antidepressants (TCAs), may modulate these molecular signals with a therapeutic purpose based on a direct antitumoral effect and beyond the TCA use to treat neuropathic pain in oncology patients. In this review, we discuss the TCAs' safety and their central effects against neuropathic pain in cancer, and the antitumoral effects of TCAs in in vitro and preclinical studies, as well as in the clinical setting. The current evidence points out that TCAs are safe and beneficial to treat neuropathic pain associated with cancer and chemotherapy, and they block different molecular pathways used by cancer cells from different locations for tumor growth and promotion. Likewise, ongoing clinical trials evaluating the antineoplastic effects of TCAs are discussed. TCAs are very biologically active compounds, and their repurposing as antitumoral drugs is a promising and straightforward approach to treat specific cancer subtypes and to further define their molecular targets, as well as an interesting starting point to design analogues with increased antitumoral activity.

Prognostic and Predictive Effects of Tumor and Plasma miR-200c-3p in Locally Advanced and Metastatic Breast Cancer.

While the role of miR-200c in cancer progression has been established, its expression and prognostic role in breast cancer is not completely understood. The predictive role of miR-200c in response to chemotherapy has also been suggested by some studies, but only limited clinical evidence is available. The purpose of this study was to investigate miR-200c-3p in the plasma and primary tumor of BC patients. The study design included two cohorts involving women with locally advanced (LABC) and metastatic breast cancer. Tumor and plasma samples were obtained before and after treatment. We found that miR-200c-3p was significantly higher in the plasma of BC patients compared with the controls. No correlation of age with plasma miR-200c-3p was found for controls or for BC patients. MiR-200c-3p tumor expression was also associated with poor overall survival in LABC patients treated with neoadjuvant chemotherapy, independently of pathological complete response or clinical stage. Our findings suggest that plasmatic miR-200c-3p levels could be useful for BC staging, while the tumor expression of miR-200c-3p might provide further prognostic information beyond residual disease in BC treated with neoadjuvant chemotherapy.

Identification of Thrombosis-Related Genes in Patients with Advanced Gastric Cancer: Data from AGAMENON-SEOM Registry.

Advanced gastric cancer is one of the most thrombogenic neoplasms. However, genetic mechanisms underlying this complication remain obscure, and the molecular and histological heterogeneity of this neoplasm hinder the identification of thrombotic biomarkers. Therefore, our main objective was to identify genes related to thrombosis regardless of Lauren subtypes. Furthermore, in a secondary exploratory study, we seek to discover thrombosis-associated genes that were specific to each TCGA molecular subtype. We designed a nested case-control study using the cohort of the AGAMENON national advanced gastric cancer registry. Ninety-seven patients were selected-48 with and 49 without venous thromboembolism (using propensity score matching to adjust for confounding factors)-and a differential gene expression array stratified by Lauren histopathological subtypes was carried out in primary tumor samples. For the secondary objective, the aforementioned differential expression analysis was conducted for each TCGA group. Fifteen genes were determined to be associated with thrombosis with the same expression trend in both the intestinal and diffuse subtypes. In thrombotic subjects, CRELD1, KCNH8, CRYGN, MAGEB16, SAA1, ARL11, CCDC169, TRMT61A, RIPPLY3 and PLA2G6 were underexpressed (adjusted-p < 0.05), while PRKD3, MIR5683, SDCBP, EPS8 and CDC45 were overexpressed (adjusted-p < 0.05), and correlated, by logistic regression, with lower or higher thrombotic risk, respectively, in the overall cohort. In each TCGA molecular subtype, we identified a series of genes differentially expressed in thrombosis that appear to be subtype-specific. We have identified several genes associated with venous thromboembolism in advanced gastric cancer that are common to Lauren intestinal and diffuse subtypes. Should these genetic factors be validated in the future, they could be complemented with existing clinical models to bolster the ability to predict thrombotic risk in individuals with advanced gastric adenocarcinoma.

Pleiotropic Effects of the P5-Type ATPase SpfA on Stress Response Networks Contribute to Virulence in the Pathogenic Mold Aspergillus fumigatus.

Aspergillus fumigatus is a human-pathogenic mold that extracts nutrients from the environment or from host tissues by secreting hydrolytic enzymes. The ability of A. fumigatus to adjust secretion levels in proportion to demand relies on the assistance of the unfolded protein response (UPR), an adaptive stress response pathway that regulates the unique protein-folding environment of the endoplasmic reticulum (ER). The P5-type ATPase Spf1 has recently been implicated in a novel mechanism of ER homeostasis that involves correcting errors in ER-membrane protein targeting. However, the contribution of this protein to the biology of A. fumigatus is unknown. Here, we employed a gene knockout and RNA sequencing strategy to determine the functional role of the A. fumigatus gene coding for the orthologous P5 ATPase SpfA. The data reveal that the spfA gene is induced by ER stress in a UPR-dependent manner. In the absence of spfA, the A. fumigatus transcriptome shifts toward a profile of altered redox and lipid balance, in addition to a signature of ER stress that includes srcA, encoding a second P-type ATPase in the ER. A ΔspfA deletion mutant showed increased sensitivity to ER stress, oxidative stress, and antifungal drugs that target the cell wall or plasma membrane. The combined loss of spfA and srcA exacerbated these phenotypes and attenuated virulence in two animal infection models. These findings demonstrate that the ER-resident ATPases SpfA and SrcA act jointly to support diverse adaptive functions of the ER that are necessary for fitness in the host environment. IMPORTANCE The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus.

Anti-Tumor Functions of Prelatent Antithrombin on Glioblastoma Multiforme Cells.

Antithrombin, the main physiological inhibitor of the coagulation cascade, exerts anti-tumor effects on glioblastoma multiforme cells. Antithrombin has different conformations: native, heparin-activated, prelatent, latent, and cleaved. The prelatent form has an intermediate affinity between latent and native antithrombin, although it is the most antiangiogenic form. Herein, we investigate the effect of this conformation on the tumorigenic processes of glioblastoma multiforme cells. Antithrombin forms were purified by chromatography. Chromogenic/fluorogenic assays were carried out to evaluate enteropeptidase and hepsin inhibition, two serine proteases involved in these processes. Wound healing, Matrigel invasion and BrdU incorporation assays were performed to study migration, invasion and proliferation. E-cadherin, Vimentin, VEGFA, pAKT, STAT3, pSTAT3, and pERK1/2 expression was assessed by Western blot and/or qRT-PCR. Prelatent antithrombin inhibited both enteropeptidase and hepsin, although it was less efficient than the native conformation. Exposure to prelatent antithrombin significantly reduced migration and invasion but not proliferation of U-87 MG, being the conformation most efficient on migration. Prelatent antithrombin down-regulated VEGFA, pSTAT3, and pERK1/2 expression in U-87 MG cells. Our work elucidates that prelatent antithrombin has surprisingly versatile anti-tumor properties in U-87 MG glioblastoma multiforme cells. This associates with resistance pathway activation, the decreased expression of tumorigenic proteins, and increased angiogenesis, postulating the existence of a new, formerly unknown receptor with potential therapeutic implications.

First exploratory study on the metabolome from plasma exosomes in patients with paroxysmal nocturnal hemoglobinuria.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease in which patients are at increased risk of thrombosis. The mechanisms underlying the associated thrombosis risk are still poorly understood, although it is known that Eculizumab, the drug of choice for symptomatic patients, prevents thrombotic events. Exosomes are extracellular vesicles that can carry and disseminate genetic material, tumor biomarkers and inflammatory mediators. To date, the metabolite cargo of plasma exosomes from PNH patients has not yet been explored. In this pilot trial, we compared the metabolome of plasma exosomes from PNH patients with that of healthy subjects in order to provide further insights into this rare disease. RESULTS: We used a non-targeted metabolomics approach with UPLC-ESI-QTOF-MS/MS and GC-MS platforms. Multivariate analyses revealed the differential occurrence (p < .001) of 78 metabolites in plasma exosomes from PNH patients vs healthy control subjects. Remarkably, prostaglandin F2-alpha (6.1-fold), stearoyl arginine (5.3-fold) and 26-hydroxycholesterol-3-sulfate (11.2-fold) were higher in PNH patients vs healthy controls (p < .001). CONCLUSIONS: This is the first description on the differential metabolite cargo occurring in plasma exosomes from PNH patients. Our results could contribute to the search for possible prognostic biomarkers of thrombotic risk in patients with PNH. Further research in a larger cohort to validate these results is warranted.

Antithrombin is incorporated into exosomes produced by antithrombin non-expressing cells.

Antithrombin is a serine protease inhibitor that exerts a crucial role in hemostasis as the main inhibitor of the coagulation cascade. It plays also critical roles in other processes, such as inflammation and cancer. Here we show that exosomes released by Madin-Darby canine kidney (MDCK) cells cultured in the presence of heparin incorporate antithrombin from the serum. Exosomal antithrombin is found complexed with the serine protease high temperature requirement A1 (HTRA1), whose cellular levels are increased after serum deprival, the condition used to collect exosomes. Although the biological relevance of the presence of antithrombin in exosomes remains to be investigated, our results suggest a functional interplay between antithrombin and HTRA1.