120 results on '"Alessandra Eva"'
Search Results
2. Proteomic profiling of extracellular vesicles in synovial fluid and plasma from Oligoarticular Juvenile Idiopathic Arthritis patients reveals novel immunopathogenic biomarkers
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Federica Raggi, Martina Bartolucci, Davide Cangelosi, Chiara Rossi, Simone Pelassa, Chiara Trincianti, Andrea Petretto, Giovanni Filocamo, Adele Civino, Alessandra Eva, Angelo Ravelli, Alessandro Consolaro, and Maria Carla Bosco
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oligoarticular juvenile idiopathic arthritis ,proteomics ,extracellular vesicles ,biomarkers ,inflammatory and immune processes ,Immunologic diseases. Allergy ,RC581-607 - Abstract
IntroductionNew early low-invasive biomarkers are demanded for the management of Oligoarticular Juvenile Idiopathic Arthritis (OJIA), the most common chronic pediatric rheumatic disease in Western countries and a leading cause of disability. A deeper understanding of the molecular basis of OJIA pathophysiology is essential for identifying new biomarkers for earlier disease diagnosis and patient stratification and to guide targeted therapeutic intervention. Proteomic profiling of extracellular vesicles (EVs) released in biological fluids has recently emerged as a minimally invasive approach to elucidate adult arthritis pathogenic mechanisms and identify new biomarkers. However, EV-prot expression and potential as biomarkers in OJIA have not been explored. This study represents the first detailed longitudinal characterization of the EV-proteome in OJIA patients.MethodsFourty-five OJIA patients were recruited at disease onset and followed up for 24 months, and protein expression profiling was carried out by liquid chromatography-tandem mass spectrometry in EVs isolated from plasma (PL) and synovial fluid (SF) samples.ResultsWe first compared the EV-proteome of SF vs paired PL and identified a panel of EV-prots whose expression was significantly deregulated in SF. Interaction network and GO enrichment analyses performed on deregulated EV-prots through STRING database and ShinyGO webserver revealed enrichment in processes related to cartilage/bone metabolism and inflammation, suggesting their role in OJIA pathogenesis and potential value as early molecular indicators of OJIA development. Comparative analysis of the EV-proteome in PL and SF from OJIA patients vs PL from age/gender-matched control children was then carried out. We detected altered expression of a panel of EV-prots able to differentiate new-onset OJIA patients from control children, potentially representing a disease-associated signature measurable at both the systemic and local levels with diagnostic potential. Deregulated EV-prots were significantly associated with biological processes related to innate immunity, antigen processing and presentation, and cytoskeleton organization. Finally, we ran WGCNA on the SF- and PL-derived EV-prot datasets and identified a few EV-prot modules associated with different clinical parameters stratifying OJIA patients in distinct subgroups.DiscussionThese data provide novel mechanistic insights into OJIA pathophysiology and an important contribution in the search of new candidate molecular biomarkers for the disease.
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- 2023
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3. Plasma-Derived Exosome Proteins as Novel Diagnostic and Prognostic Biomarkers in Neuroblastoma Patients
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Martina Morini, Federica Raggi, Martina Bartolucci, Andrea Petretto, Martina Ardito, Chiara Rossi, Daniela Segalerba, Alberto Garaventa, Alessandra Eva, Davide Cangelosi, and Maria Carla Bosco
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neuroblastoma ,exosomes ,biomarkers ,Cytology ,QH573-671 - Abstract
Neuroblastoma (NB) is the most common extracranial solid tumor during infancy, causing up to 10% of mortality in children; thus, identifying novel early and accurate diagnostic and prognostic biomarkers is mandatory. NB-derived exosomes carry proteins (Exo-prots) reflecting the status of the tumor cell of origin. The purpose of this study was to characterize, for the first time, the Exo-prots specifically expressed in NB patients associated with tumor phenotype and disease stage. We isolated exosomes from plasma specimens of 24 HR-NB patients and 24 low-risk (LR-NB) patients at diagnosis and of 24 age-matched healthy controls (CTRL). Exo-prot expression was measured by liquid chromatography–mass spectrometry. The data are available via ProteomeXchange (PXD042422). The NB patients had a different Exo-prot expression profile compared to the CTRL. The deregulated Exo-prots in the NB specimens acted mainly in the tumor-associated pathways. The HR-NB patients showed a different Exo-prot expression profile compared to the LR-NB patients, with the modulation of proteins involved in cell migration, proliferation and metastasis. NCAM, NCL, LUM and VASP demonstrated a diagnostic value in discriminating the NB patients from the CTRL; meanwhile, MYH9, FN1, CALR, AKAP12 and LTBP1 were able to differentiate between the HR-NB and LR-NB patients with high accuracy. Therefore, Exo-prots contribute to NB tumor development and to the aggressive metastatic NB phenotype.
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- 2023
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4. Nucleolin expression has prognostic value in neuroblastoma patients
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Davide Cangelosi, Chiara Brignole, Veronica Bensa, Roberto Tamma, Fabiana Malaguti, Barbara Carlini, Elena Giusto, Enzo Calarco, Patrizia Perri, Domenico Ribatti, Nuno André Fonseca, Joao Nuno Moreira, Alessandra Eva, Loredana Amoroso, Massimo Conte, Alberto Garaventa, Angela Rita Sementa, Maria Valeria Corrias, Mirco Ponzoni, and Fabio Pastorino
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Neuroblastoma ,Nucleolin ,Biomarker ,Prognostic value ,Medicine ,Medicine (General) ,R5-920 - Abstract
Summary: Background: Neuroblastoma (NB) represents the most frequent form of extra-cranial solid tumour of infants, responsible for 15% of childhood cancer deaths. Nucleolin (NCL) prognostic value in NB was investigated. Methods: NCL protein expression was retrospectively evaluated in tumour samples of NB patients at diagnosis and after chemotherapy. NCL prognostic value at mRNA level was assessed in a cohort of 20 patients with stage 4 NB (qPCR20, n=20, discovery dataset) and in the MultiPlatform786 including 786 patients of all stages (validation dataset). Overall and event-free survival curves were plotted by Kaplan-Meier method and compared by log-rank test. Findings: NCL protein, down-modulated after chemotherapy in association with features of neuroblastic differentiation,resulted statistically significantly overexpressed in NB tumours and higher in stage 4 compared to stage 1,2,3 patients. In the stage 4 patients cohort qPCR20, patients with high NCLmRNA expression revealed a statisticallysignificant lower survival probability than those with low NCL expression (OS: HR 4.1 95%CI 1.2–13.8;p=0.0215[Log-rank test], EFS: HR 4.1 95%CI 1.2–14.0, p=0.0197[Log-rank test]). In the MultiPlatform786 (n=786), multivariate analysis suggested thatNCL expression has a statistically significant prognostic value even in the model adjusted for established prognostic markers. NCL expression significantly stratified also patients with >18 months and stage 4 tumour (OS: HR 1.8 95%CI 1.2–2.7, p=0.0009[Log-rank test]; EFS: HR 1.7 95%CI 1.1–2.5, p=0.002[Log-rank test]), patients with>18 months stage 4 with MYCN non amplified tumour[EFS: HR 2.3 95%CI 1.2–4.7, p=0.01[Log-rank test]), and patients with MYCN non amplified and MYC high [OS: HR 11.9 95%CI 2.3–62.4, p=0.003[Log-rank test]; EFS: HR 7.2 95%CI 1.6–33.4, p=0.01[Log-rank test]). A statistically significant correlation between NCL and MYCN, MYC, and TERT was found in independent datasets (MultiPlatform786 (n=786) and Agilent394 (n=394). Gene set enrichment analysis revealed a statisticallysignificant positive enrichment of MYC target genes and genes involved in telomerase maintenance. Interpretation: NCL is a novel and independent (adjusting for age, INSS stage, and MYCN status) prognostic marker for NB. Funding: IMH-EuroNanoMed II-2015 and AIRC-IG.
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- 2022
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5. Extracellular vesicle‐derived microRNAs as potential biomarkers in oligoarticular juvenile idiopathic arthritis patients: methodological challenges and new perspectives
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Federica Raggi, Davide Cangelosi, Alessandro Consolaro, Chiara Rossi, Simone Pelassa, Katia Cortese, Maria Cristina Gagliani, Martina Morini, Daniela Segalerba, Chiara Brignole, Paola Bocca, Danilo Marimpietri, Chiara Trincianti, Angelo Ravelli, Alessandra Eva, and Maria Carla Bosco
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Medicine (General) ,R5-920 - Published
- 2022
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6. Untargeted LC-HRMS Based-Plasma Metabolomics Reveals 3-O-Methyldopa as a New Biomarker of Poor Prognosis in High-Risk Neuroblastoma
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Sebastiano Barco, Chiara Lavarello, Davide Cangelosi, Martina Morini, Alessandra Eva, Luca Oneto, Paolo Uva, Gino Tripodi, Alberto Garaventa, Massimo Conte, Andrea Petretto, and Giuliana Cangemi
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neuroblastoma ,metabolomics ,biomarker ,high resolution mass spectrometry ,catecholamines ,3-O-methyldopa ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Neuroblastoma (NB) is the most common extracranial malignant tumor in children. Although the survival rate of NB has improved over the years, the outcome of NB still remains poor for over 30% of cases. A more accurate risk stratification remains a key point in the study of NB and the availability of novel prognostic biomarkers of “high-risk” at diagnosis could help improving patient stratification and predicting outcome.In this paper we show a biomarker discovery approach applied to the plasma of 172 NB patients. Plasma samples from a first cohort of NB patients and age-matched healthy controls were used for untargeted metabolomics analysis based on high-resolution mass spectrometry (HRMS). Differential expression analysis highlighted a number of metabolites annotated with a high degree of identification. Among them, 3-O-methyldopa (3-O-MD) was validated in a second cohort of NB patients using a targeted metabolite profiling approach and its prognostic potential was also analyzed by survival analysis on patients with 3 years follow-up. High expression of 3-O-MD was associated with worse prognosis in the subset of patients with stage M tumor (log-rank p < 0.05) and, among them, it was confirmed as a prognostic factor able to stratify high-risk patients older than 18 months. 3-O-MD might be thus considered as a novel prognostic biomarker of NB eligible to be included at diagnosis among catecholamine metabolite panels in prospective clinical studies. Further studies are warranted to exploit other potential biomarkers highlighted using our approach.
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- 2022
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7. Transcriptome analysis defines myocardium gene signatures in children with ToF and ASD and reveals disease-specific molecular reprogramming in response to surgery with cardiopulmonary bypass
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Federica Raggi, Davide Cangelosi, Pamela Becherini, Fabiola Blengio, Martina Morini, Massimo Acquaviva, Maria Luisa Belli, Giuseppe Panizzon, Giuseppe Cervo, Luigi Varesio, Alessandra Eva, and Maria Carla Bosco
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Gene expression profiling ,Congenital heart disease ,Cardiopulmonary bypass ,Atrial myocardium ,Hypoxia ,Medicine - Abstract
Abstract Background Tetralogy of Fallot (ToF) and Atrial Septal Defects (ASD) are the most common types of congenital heart diseases and a major cause of childhood morbidity and mortality. Cardiopulmonary bypass (CPB) is used during corrective cardiac surgery to support circulation and heart stabilization. However, this procedure triggers systemic inflammatory and stress response and consequent increased risk of postoperative complications. The aim of this study was to define the molecular bases of ToF and ASD pathogenesis and response to CPB and identify new potential biomarkers. Methods Comparative transcriptome analysis of right atrium specimens collected from 10 ToF and 10 ASD patients was conducted before (Pre-CPB) and after (Post-CPB) corrective surgery. Total RNA isolated from each sample was individually hybridized on Affymetrix HG-U133 Plus Array Strips containing 38,500 unique human genes. Differences in the gene expression profiles and functional enrichment/network analyses were assessed using bioinformatic tools. qRT-PCR analysis was used to validate gene modulation. Results Pre-CPB samples showed significant differential expression of a total of 72 genes, 28 of which were overexpressed in ToF and 44 in ASD. According to Gene Ontology annotation, the mostly enriched biological processes were represented by matrix organization and cell adhesion in ToF and by muscle development and contractility in ASD specimens. GSEA highlighted the specific enrichment of hypoxia gene sets in ToF samples, pointing to a role for hypoxia in disease pathogenesis. The post-CPB myocardium exhibited significant alterations in the expression profile of genes related to transcription regulation, growth/apoptosis, inflammation, adhesion/matrix organization, and oxidative stress. Among them, only 70 were common to the two disease groups, whereas 110 and 24 were unique in ToF and ASD, respectively. Multiple functional interactions among differentially expressed gene products were predicted by network analysis. Interestingly, gene expression changes in ASD samples followed a consensus hypoxia profile. Conclusion Our results provide a comprehensive view of gene reprogramming in right atrium tissues of ToF and ASD patients before and after CPB, defining specific molecular pathways underlying disease pathophysiology and myocardium response to CPB. These findings have potential translational value because they identify new candidate prognostic markers and targets for tailored cardioprotective post-surgical therapies.
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- 2020
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8. MYC regulates metabolism through vesicular transfer of glycolytic kinases
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Alexia Tsakaneli, Victor Corasolla Carregari, Martina Morini, Alessandra Eva, Giuliana Cangemi, Olesya Chayka, Evgeny Makarov, Sandra Bibbò, Emily Capone, Gianluca Sala, Vincenzo De Laurenzi, Evon Poon, Louis Chesler, Luisa Pieroni, Martin R. Larsen, Giuseppe Palmisano, and Arturo Sala
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extracellular vesicles ,MYC ,MYCN ,neuroblastoma ,Warburg effect ,Biology (General) ,QH301-705.5 - Abstract
Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN-expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer-promoting activity of EVs to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYC-expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and hexokinase II were detected in the EVs circulating in the bloodstream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC-activated cancers might spread oncogenic signals to remote body locations through EVs.
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- 2021
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9. The SGLT2-inhibitor dapagliflozin improves neutropenia and neutrophil dysfunction in a mouse model of the inherited metabolic disorder GSDIb
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Roberta Resaz, Federica Raggi, Daniela Segalerba, Chiara Lavarello, Alessandra Gamberucci, Maria Carla Bosco, Simonetta Astigiano, Antonia Assunto, Daniela Melis, Mariavittoria D'Acierno, Maria Veiga-da-Cunha, Andrea Petretto, Paola Marcolongo, Francesco Trepiccione, and Alessandra Eva
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Glycogen storage disease type 1b ,1,5-anhydroglucitol-6-phosphate ,Neutrophils ,Dapagliflozin ,Renal sodium-glucose co-transporter-2 ,Mouse model ,Medicine (General) ,R5-920 ,Biology (General) ,QH301-705.5 - Abstract
Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells.
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- 2021
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10. Multiparametric flow cytometry highlights B7-H3 as a novel diagnostic/therapeutic target in GD2neg/low neuroblastoma variants
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Alessandra Dondero, Martina Morini, Davide Cangelosi, Katia Mazzocco, Martina Serra, Grazia Maria Spaggiari, Gianluca Rotta, Annalisa Tondo, Aurora Castellano, Francesca Scuderi, Angela Rita Sementa, Alessandra Eva, Massimo Conte, Alberto Garaventa, Cristina Bottino, and Roberta Castriconi
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Background High-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of GD2. The mechanisms responsible for therapy resistance are unclear and might include the presence of GD2neg/low NB variants and/or the expression of immune checkpoint ligands such as B7-H3.Method Here, we describe a multiparametric flow cytometry (MFC) combining the acquisition of 106 nucleated singlets, Syto16pos CD45neg CD56pos cells, and the analysis of GD2 and B7-H3 surface expression. 41 bone marrow (BM) aspirates from 25 patients with NB, at the onset or relapse, are analyzed, comparing results with cytomorphological analysis (CA) and/or immunohistochemistry (IHC). Spike in experiments assesses the sensitivity of MFC. Kaplan-Meier analysis on 498 primary NBs selects novel prognostic markers possibly integrating the MFC panel.Results No false positive are detected, and MFC shows high sensitivity (0.0005%). Optimized MFC identifies CD45negCD56pos NB cells in 11 out of 12 (91.6%) of BM indicated as infiltrated by CA, 7 of which coexpress high levels of GD2 and B7-H3. MFC detects CD45negCD56posGD2neg/low NB variants expressing high surface levels of B7-H3 in two patients with HR-NB (stage M) diagnosed at 53 and 139 months of age. One of them has a non-MYCN amplified tumor with unusual THpos PHOX2Bneg phenotype, which relapsed 141 months post-diagnosis with BM infiltration and a humerus lesion. All GD2neg/low NB variants are detected in patients at relapse. Kaplan-Meier analysis highlights an interesting dichotomous prognostic value of MML5, ULBPs, PVR, B7-H6, and CD47, ligands involved in NB recognition by the immune system.Conclusions Our study validates a sensitive MFC analysis providing information on GD2 and B7-H3 surface expression and allowing fast, specific and sensitive evaluation of BM tumor burden. With other routinely used diagnostic and prognostic tools, MFC can improve diagnosis, prognosis, orienting novel personalized treatments in patients with GD2low/neg NB, who might benefit from innovative therapies combining B7-H3 targeting.
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- 2021
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11. Circulating exosomal microRNAs as potential biomarkers of hepatic injury and inflammation in a murine model of glycogen storage disease type 1a
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Roberta Resaz, Davide Cangelosi, Martina Morini, Daniela Segalerba, Luca Mastracci, Federica Grillo, Maria Carla Bosco, Cristina Bottino, Irma Colombo, and Alessandra Eva
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glycogen storage disease type 1a ,hepatocellular adenoma ,biomarkers ,exosomes ,liver ,microrna ,Medicine ,Pathology ,RB1-214 - Abstract
Most patients affected by glycogen storage disease type 1a (GSD1a), an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-α (G6Pase-α), develop renal and liver complications, including the development of hepatocellular adenoma/carcinoma. The purpose of this study was to identify potential biomarkers of the pathophysiology of the GSD1a-affected liver. To this end, we used the plasma exosomes of a murine model of GSD1a, the LS-G6pc−/− mouse, to uncover the modulation in microRNA expression associated with the disease. The microRNAs differentially expressed between LS-G6pc−/− and wild-type mice, LS-G6pc−/− mice with hepatocellular adenoma and LS-G6pc−/− mice without adenoma, and LS-G6pc−/− mice with amyloidosis and LS-G6pc−/− mice without amyloidosis were identified. Pathway analysis demonstrated that the target genes of the differentially expressed microRNA were significantly enriched for the insulin signaling pathway, glucose and lipid metabolism, Wnt/β-catenin, telomere maintenance and hepatocellular carcinoma, and chemokine and immune regulation signaling pathways. Although some microRNAs were common to the different pathologic conditions, others were unique to the cancerous or inflammatory status of the animals. Therefore, the altered expression of several microRNAs is correlated with various pathologic liver states and might help to distinguish them during the progression of the disease and the development of late GSD1a-associated complications.
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- 2020
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12. Characterization of high- and low-risk hepatocellular adenomas by magnetic resonance imaging in an animal model of glycogen storage disease type 1A
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Roberta Resaz, Francesca Rosa, Federica Grillo, Luca Basso, Daniela Segalerba, Andrea Puglisi, Maria Carla Bosco, Luca Mastracci, Carlo E. Neumaier, Luigi Varesio, and Alessandra Eva
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Liver ,Tumor ,Magnetic resonance ,β-Catenin ,Medicine ,Pathology ,RB1-214 - Abstract
Hepatocellular adenomas (HCAs) are benign tumors, of which the most serious complications are hemorrhage and malignant transformation to hepatocellular carcinoma (HCC). Among the various subtypes of HCA, the β-catenin-activated subtype (bHCA) is associated with greatest risk of malignant transformation. Magnetic resonance imaging (MRI) is an important tool to differentiate benign and malignant hepatic lesions, and preclinical experimental approaches may help to develop a method to identify MRI features associated with bHCA. HCAs are associated with various pathologies, including glycogen storage disease 1a (GSD1a). Here, we utilized a mouse model for GSD1a that develops HCA and HCC, and analyzed the mice in order to distinguish low-risk from high-risk tumors. Animals were scanned by MRI using a hepato-specific contrast agent. The mice were sacrificed after MRI and their lesions were classified using immunohistochemistry. We observed that 45% of the animals developed focal lesions, and MRI identified four different patterns after contrast administration: isointense, hyperintense and hypointense lesions, and lesions with peripheral contrast enhancement. After contrast administration, only bHCA and HCC were hypointense in T1-weighted imaging and mildly hyperintense in T2-weighted imaging. Thus, high-risk adenomas display MRI features clearly distinguishable from those exhibited by low-risk adenomas, indicating that MRI is a reliable method for early diagnosis and classification of HCA, necessary for correct patient management.
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- 2019
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13. MCM2 and Carbonic Anhydrase 9 Are Novel Potential Targets for Neuroblastoma Pharmacological Treatment
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Patrizia Garbati, Raffaella Barbieri, Davide Cangelosi, Carlo Zanon, Delfina Costa, Alessandra Eva, Stefano Thellung, Matilde Calderoni, Francesca Baldini, Gian Paolo Tonini, Paola Modesto, Tullio Florio, and Aldo Pagano
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neuroblastoma ,carbonic anhydrase 9 ,MCM2 ,ciprofloxacin ,acetazolamide ,Biology (General) ,QH301-705.5 - Abstract
To overcome the lack of effective pharmacological treatments for high-risk neuroblastoma (HR-NB), the development of novel in vitro and in vivo models that better recapitulate the disease is required. Here, we used an in vitro multiclonal cell model encompassing NB cell differentiation stages, to identify potential novel pharmacological targets. This model allowed us to identify, by low-density RT-PCR arrays, two gene sets, one over-expressed during NB cell differentiation, and the other up-regulated in more malignant cells. Challenging two HR-NB gene expression datasets, we found that these two gene sets are related to high and low survival, respectively. Using mouse NB cisplatin-treated xenografts, we identified two genes within the list associated to the malignant stage (MCM2 and carbonic anhydrase 9), whose expression is positively correlated with tumor growth. Thus, we tested their pharmacological targeting as potential therapeutic strategy. We measured mice survival and tumor growth rate after xenografts of human NB treated with cisplatin in the presence of MCM2/carbonic anhydrase 9 inhibitors (ciprofloxacin and acetazolamide). MCM2 or carbonic anhydrase 9 inhibition significantly increased cisplatin activity, supporting their possible testing for NB therapy.
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- 2020
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14. Hypoxia Modifies the Transcriptome of Human NK Cells, Modulates Their Immunoregulatory Profile, and Influences NK Cell Subset Migration
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Monica Parodi, Federica Raggi, Davide Cangelosi, Claudia Manzini, Mirna Balsamo, Fabiola Blengio, Alessandra Eva, Luigi Varesio, Gabriella Pietra, Lorenzo Moretta, Maria Cristina Mingari, Massimo Vitale, and Maria Carla Bosco
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NK cells ,hypoxia ,tumor immunology ,cytokines/chemokines ,chemokine receptors ,CD56bright cells ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Hypoxia, which characterizes most tumor tissues, can alter the function of different immune cell types, favoring tumor escape mechanisms. In this study, we show that hypoxia profoundly acts on NK cells by influencing their transcriptome, affecting their immunoregulatory functions, and changing the chemotactic responses of different NK cell subsets. Exposure of human peripheral blood NK cells to hypoxia for 16 or 96 h caused significant changes in the expression of 729 or 1,100 genes, respectively. Gene Set Enrichment Analysis demonstrated that these changes followed a consensus hypoxia transcriptional profile. As assessed by Gene Ontology annotation, hypoxia-targeted genes were implicated in several biological processes: metabolism, cell cycle, differentiation, apoptosis, cell stress, and cytoskeleton organization. The hypoxic transcriptome also showed changes in genes with immunological relevance including those coding for proinflammatory cytokines, chemokines, and chemokine-receptors. Quantitative RT-PCR analysis confirmed the modulation of several immune-related genes, prompting further immunophenotypic and functional studies. Multiplex ELISA demonstrated that hypoxia could variably reduce NK cell ability to release IFNγ, TNFα, GM-CSF, CCL3, and CCL5 following PMA+Ionomycin or IL15+IL18 stimulation, while it poorly affected the response to IL12+IL18. Cytofluorimetric analysis showed that hypoxia could influence NK chemokine receptor pattern by sustaining the expression of CCR7 and CXCR4. Remarkably, this effect occurred selectively (CCR7) or preferentially (CXCR4) on CD56bright NK cells, which indeed showed higher chemotaxis to CCL19, CCL21, or CXCL12. Collectively, our data suggest that the hypoxic environment may profoundly influence the nature of the NK cell infiltrate and its effects on immune-mediated responses within tumor tissues.
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- 2018
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15. Regulation of Human Macrophage M1–M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1
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Federica Raggi, Simone Pelassa, Daniele Pierobon, Federica Penco, Marco Gattorno, Francesco Novelli, Alessandra Eva, Luigi Varesio, Mirella Giovarelli, and Maria Carla Bosco
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macrophages ,hypoxia ,polarization ,immunoregulatory receptors ,inflammation ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Macrophages (Mf) are a heterogeneous population of tissue-resident professional phagocytes and a major component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth. They can undergo diverse forms of activation in response to environmental factors, polarizing into specialized functional subsets. A common hallmark of the pathologic environment is represented by hypoxia. The impact of hypoxia on human Mf polarization has not been fully established. The objective of this study was to elucidate the effects of a hypoxic environment reflecting that occurring in vivo in diseased tissues on the ability of human Mf to polarize into classically activated (proinflammatory M1) and alternatively activated (anti-inflammatory M2) subsets. We present data showing that hypoxia hinders Mf polarization toward the M1 phenotype by decreasing the expression of T cell costimulatory molecules and chemokine homing receptors and the production of proinflammatory, Th1-priming cytokines typical of classical activation, while promoting their acquisition of phenotypic and secretory features of alternative activation. Furthermore, we identify the triggering receptor expressed on myeloid cells (TREM)-1, a member of the Ig-like immunoregulatory receptor family, as a hypoxia-inducible gene in Mf and demonstrate that its engagement by an agonist Ab reverses the M2-polarizing effect of hypoxia imparting a M1-skewed phenotype to Mf. Finally, we provide evidence that Mf infiltrating the inflamed hypoxic joints of children affected by oligoarticular juvenile idiopatic arthritis express high surface levels of TREM-1 associated with predominant M1 polarization and suggest the potential of this molecule in driving M1 proinflammatory reprogramming in the hypoxic synovial environment.
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- 2017
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16. Immunohistochemical analysis of PDK1, PHD3 and HIF-1α expression defines the hypoxic status of neuroblastoma tumors.
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Marzia Ognibene, Davide Cangelosi, Martina Morini, Daniela Segalerba, Maria Carla Bosco, Angela Rita Sementa, Alessandra Eva, and Luigi Varesio
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Medicine ,Science - Abstract
Neuroblastoma (NB) is the most common solid tumor during infancy and the first cause of death among the preschool age diseases. The availability of several NB genomic profiles improves the prognostic ability, but the outcome prediction for this pathology remains imperfect. We previously produced a novel prognostic gene signature based on the response of NB cells to hypoxia, a condition of tumor microenvironment strictly connected with cancer aggressiveness. Here we attempted to further define the expression of hypoxia-modulated specific genes, looking at their protein level in NB specimens, considering in particular the hypoxia inducible factor-1α (HIF-1α), the mitochondrial pyruvate dehydrogenase kinase 1 (PDK1), and the HIF-prolyl hydroxylase domain 3 (PHD3). The evaluation of expression was performed by Western blot and immunocytochemistry on NB cell lines and by immunohistochemistry on tumor specimens. Stimulation of both HIF-1α and PDK1 and inhibition of PHD3 expression were observed in NB cell lines cultured under prolonged hypoxic conditions as well as in most of the tumors with poor outcome. Our results indicate that the immunohistochemistry analysis of the protein expression of PDK1, PHD3, and HIF-1α defines the hypoxic status of NB tumors and can be used as a simple and relevant tool to stratify high-risk patients.
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- 2017
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17. Development of hepatocellular adenomas and carcinomas in mice with liver-specific G6Pase-α deficiency
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Roberta Resaz, Cristina Vanni, Daniela Segalerba, Angela R. Sementa, Luca Mastracci, Federica Grillo, Daniele Murgia, Maria Carla Bosco, Janice Y. Chou, Ottavia Barbieri, Luigi Varesio, and Alessandra Eva
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Glycogen storage disease type 1a ,Glucose-6-phosphatase-α ,Animal model ,Hepatomegaly ,Hepatic steatosis ,Hepatocellular adenoma ,Hepatocellular carcinoma ,Medicine ,Pathology ,RB1-214 - Abstract
Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α), and is characterized by impaired glucose homeostasis and a high risk of developing hepatocellular adenomas (HCAs). A globally G6Pase-α-deficient (G6pc−/−) mouse model that shows pathological features similar to those of humans with GSD-1a has been developed. These mice show a very severe phenotype of disturbed glucose homeostasis and rarely live beyond weaning. We generated liver-specific G6Pase-α-deficient (LS‑G6pc−/−) mice as an alternative animal model for studying the long-term pathophysiology of the liver and the potential treatment strategies, such as cell therapy. LS‑G6pc−/− mice were viable and exhibited normal glucose profiles in the fed state, but showed significantly lower blood glucose levels than their control littermates after 6 hours of fasting. LS‑G6pc−/− mice developed hepatomegaly with glycogen accumulation and hepatic steatosis, and progressive hepatic degeneration. Ninety percent of the mice analyzed developed amyloidosis by 12 months of age. Finally, 25% of the mice sacrificed at age 10–20 months showed the presence of multiple HCAs and in one case late development of hepatocellular carcinoma (HCC). In conclusion, LS‑G6pc−/− mice manifest hepatic symptoms similar to those of human GSD-1a and, therefore, represent a valid model to evaluate long-term liver pathogenesis of GSD-1a.
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- 2014
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18. Induction of epithelial mesenchimal transition and vasculogenesis in the lenses of Dbl oncogene transgenic mice.
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Paolo Fardin, Marzia Ognibene, Cristina Vanni, Amleto De Santanna, Luigi Varesio, and Alessandra Eva
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Medicine ,Science - Abstract
BACKGROUND: The Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. The numerous domains that are present in many Dbl family proteins suggest that they act to integrate multiple inputs in complicated signaling networks involving the Rho GTPases. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We generated transgenic mice introducing the cDNA of Dbl oncogene linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression in mouse lenses affected proliferation, migration and differentiation of lens epithelial cells. RESULTS: We used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of 2 days, 2 weeks, and 6 weeks old transgenic mice. We observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT), such as down-regulation of epithelial cell markers and up-regulation of fibroblast markers. Genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses where vascularization can be readily observed. CONCLUSION: Onco-Dbl expression in mouse lens correlated with modulation of genes involved in the regulation of EMT, apoptosis and vasculogenesis leading to disruption of the lens architecture, epithelial cell proliferation, and aberrant angiogenesis. We conclude that onco-Dbl has a potentially important, previously unreported, capacity to dramatically alter epithelial cell migration, replication, polarization and differentiation and to induce vascularization of an epithelial tissue.
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- 2009
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19. Dapagliflozin Prevents Kidney Glycogen Accumulation and Improves Renal Proximal Tubule Cell Functions in a Mouse Model of Glycogen Storage Disease Type 1b
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Mariavittoria D’Acierno, Roberta Resaz, Anna Iervolino, Rikke Nielsen, Donato Sardella, Sabrina Siccardi, Vincenzo Costanzo, Luciano D’Apolito, Yoko Suzumoto, Daniela Segalerba, Simonetta Astigiano, Alessandra F. Perna, Giovambattista Capasso, Alessandra Eva, Francesco Trepiccione, D'Acierno, Mariavittoria, Resaz, Roberta, Iervolino, Anna, Nielsen, Rikke, Sardella, Donato, Siccardi, Sabrina, Costanzo, Vincenzo, D'Apolito, Luciano, Suzumoto, Yoko, Segalerba, Daniela, Astigiano, Simonetta, Perna, Alessandra, Capasso, Giovambattista, Eva, Alessandra, and Trepiccione, Francesco
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Nephrology ,General Medicine - Abstract
BACKGROUND: Mutations in SLC37A4, which encodes the intracellular glucose transporter G6PT, cause the rare glycogen storage disease type 1b (GSD1b). A long-term consequence of GSD1b is kidney failure, which requires KRT. The main protein markers of proximal tubule function, including NaPi2A, NHE3, SGLT2, GLUT2, and AQP1, are downregulated as part of the disease phenotype.METHODS: We utilized an inducible mouse model of GSD1b, TM-G6PT-/-, to show that glycogen accumulation plays a crucial role in altering proximal tubule morphology and function. To limit glucose entry into proximal tubule cells and thus to prevent glycogen accumulation, we administered an SGLT2-inhibitor, dapagliflozin, to TM-G6PT-/- mice.RESULTS: In proximal tubule cells, G6PT suppression stimulates the upregulation and activity of hexokinase-I, which increases availability of the reabsorbed glucose for intracellular metabolism. Dapagliflozin prevented glycogen accumulation and improved kidney morphology by promoting a metabolic switch from glycogen synthesis toward lysis and by restoring expression levels of the main proximal tubule functional markers.CONCLUSION: We provide proof of concept for the efficacy of dapagliflozin in preserving kidney function in GSD1b mice. Our findings could represent the basis for repurposing this drug to treat patients with GSD1b.
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- 2022
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20. Molecular mechanism underlying impaired hepatic autophagy in glycogen storage disease type Ib
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Sudeep, Gautam, Lisa, Zhang, Cheol, Lee, Irina, Arnaoutova, Hung Dar, Chen, Roberta, Resaz, Alessandra, Eva, Brian C, Mansfield, and Janice Y, Chou
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Genetics ,Original Article ,General Medicine ,Molecular Biology ,Genetics (clinical) - Abstract
Type Ib glycogen storage disease (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that translocates G6P from the cytoplasm into the endoplasmic reticulum lumen, where the intraluminal G6P is hydrolyzed to glucose by glucose-6-phosphatase-α (G6Pase-α). Clinically, GSD-Ib patients manifest a metabolic phenotype of impaired blood glucose homeostasis and a long-term risk of hepatocellular adenoma/carcinoma (HCA/HCC). Studies have shown that autophagy deficiency contributes to hepatocarcinogenesis. In this study, we show that G6PT deficiency leads to impaired hepatic autophagy evident from attenuated expression of many components of the autophagy network, decreased autophagosome formation and reduced autophagy flux. The G6PT-deficient liver displayed impaired sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK) signaling, along with reduced expression of SIRT1, forkhead boxO3a (FoxO3a), liver kinase B-1 (LKB1) and the active p-AMPK. Importantly, we show that overexpression of either SIRT1 or LKB1 in G6PT-deficient liver restored autophagy and SIRT1/FoxO3a and LKB1/AMPK signaling. The hepatosteatosis in G6PT-deficient liver decreased SIRT1 expression. LKB1 overexpression reduced hepatic triglyceride levels, providing a potential link between LKB1/AMPK signaling upregulation and the increase in SIRT1 expression. In conclusion, downregulation of SIRT1/FoxO3a and LKB1/AMPK signaling underlies impaired hepatic autophagy which may contribute to HCA/HCC development in GSD-Ib. Understanding this mechanism may guide future therapies.
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- 2022
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21. Exosomal MicroRNAs as Potential Biomarkers of Hepatic Injury and Kidney Disease in Glycogen Storage Disease Type Ia Patients
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Roberta Resaz, Davide Cangelosi, Daniela Segalerba, Martina Morini, Paolo Uva, Maria Carla Bosco, Giuseppe Banderali, Ana Estrella, Corbinian Wanner, David A. Weinstein, Annalisa Sechi, Sabrina Paci, Daniela Melis, Maja Di Rocco, Young Mok Lee, and Alessandra Eva
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Adult ,Male ,kidney ,Time Factors ,Adolescent ,QH301-705.5 ,hepatocellular adenoma ,exosomes ,Glycogen Storage Disease Type I ,liver ,Catalysis ,Article ,Inorganic Chemistry ,Cohort Studies ,Mice ,Young Adult ,Animals ,Humans ,microRNA ,GSDIa ,biomarkers ,Physical and Theoretical Chemistry ,Biology (General) ,Child ,Preschool ,Molecular Biology ,QD1-999 ,Spectroscopy ,Gene Expression Profiling ,Organic Chemistry ,Age Factors ,General Medicine ,Middle Aged ,Computer Science Applications ,MicroRNAs ,Chemistry ,Gene Ontology ,Gene Expression Regulation ,Child, Preschool ,Case-Control Studies ,Glucose-6-Phosphatase ,Female ,Kidney Diseases ,Biomarkers ,Exosomes ,Liver - Abstract
Glycogen storage disease type Ia (GSDIa) is an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-α (G6Pase-α). Affected individuals develop renal and liver complications, including the development of hepatocellular adenoma/carcinoma and kidney failure. The purpose of this study was to identify potential biomarkers of the evolution of the disease in GSDIa patients. To this end, we analyzed the expression of exosomal microRNAs (Exo-miRs) in the plasma exosomes of 45 patients aged 6 to 63 years. Plasma from age-matched normal individuals were used as controls. We found that the altered expression of several Exo-miRs correlates with the pathologic state of the patients and might help to monitor the progression of the disease and the development of late GSDIa-associated complications.
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- 2022
22. Targeting of Ubiquitin E3 Ligase RNF5 as a Novel Therapeutic Strategy in Neuroectodermal Tumors
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Elisa Principi, Elvira Sondo, Giovanna Bianchi, Silvia Ravera, Martina Morini, Valeria Tomati, Cristina Pastorino, Federico Zara, Claudio Bruno, Alessandra Eva, Nicoletta Pedemonte, and Lizzia Raffaghello
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Cancer Research ,neuroblastoma ,Oncology ,RNF5 ,melanoma ,ubiquitin ligase ,endoplasmic reticulum associated protein degradation ,neoplasms - Abstract
RNF5, an endoplasmic reticulum (ER) E3 ubiquitin ligase, participates to the ER-associated protein degradation guaranteeing the protein homeostasis. Depending on tumor model tested, RNF5 exerts pro- or anti-tumor activity. The aim of this study was to elucidate the controversial role of RNF5 in neuroblastoma and melanoma, two neuroectodermal tumors of infancy and adulthood, respectively. RNF5 gene levels are evaluated in publicly available datasets reporting the gene expression profile of melanoma and neuroblastoma primary tumors at diagnosis. The therapeutic effect of Analog-1, an RNF5 pharmacological activator, was investigated on in vitro and in vivo neuroblastoma and melanoma models. In both neuroblastoma and melanoma patients the high expression of RNF5 correlated with a better prognostic outcome. Treatment of neuroblastoma and melanoma cell lines with Analog-1 reduced cell viability by impairing the glutamine availability and energy metabolism through inhibition of F1Fo ATP-synthase activity. This latter event led to a marked increase in oxidative stress, which, in turn, caused cell death. Similarly, neuroblastoma- and melanoma-bearing mice treated with Analog-1 showed a significant delay of tumor growth in comparison to those treated with vehicle only. These findings validate RNF5 as an innovative drug target and support the development of Analog-1 in early phase clinical trials for neuroblastoma and melanoma patients.
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- 2022
23. MYC regulates metabolism through vesicular transfer of glycolytic kinases
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Luisa Pieroni, Giuseppe Palmisano, Gianluca Sala, Martina Morini, Evon Poon, Arturo Sala, Louis Chesler, Alessandra Eva, Alexia Tsakaneli, Vincenzo De Laurenzi, Evgeny M. Makarov, Martin R. Larsen, Giuliana Cangemi, Sandra Bibbò, Emily Capone, Olesya Chayka, and Victor Corasolla Carregari
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Proteomics ,Thyroid Hormones ,QH301-705.5 ,Immunology ,MYC ,PKM2 ,Biology ,Extracellular vesicles ,General Biochemistry, Genetics and Molecular Biology ,Mass Spectrometry ,Extracellular Vesicles ,neuroblastoma ,Neuroblastoma ,Cell Line, Tumor ,Hexokinase ,MYCN ,medicine ,Humans ,Glycolysis ,Gene Regulatory Networks ,Biology (General) ,Phosphorylation ,Child ,neoplasms ,Research Articles ,Cell Proliferation ,N-Myc Proto-Oncogene Protein ,Kinase ,General Neuroscience ,CÉLULAS CULTIVADAS DE TUMOR ,Research ,Gene Amplification ,Membrane Proteins ,Metabolism ,medicine.disease ,Warburg effect ,Gene Expression Regulation, Neoplastic ,Cancer research ,Carrier Proteins ,extracellular vesicles ,Pyruvate kinase - Abstract
Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.5713034. Copyright © 2021 The Authors. Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN-expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer-promoting activity of EVs to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYC-expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and hexokinase II were detected in the EVs circulating in the bloodstream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC-activated cancers might spread oncogenic signals to remote body locations through EVs. Neuroblastoma UK to AS and FAPESP SPRINT Award (50356-4); FAPESP (2014/06863-3, 2018/18257-1, 2018/15549-1, 16/50356-4); CNPq “bolsa de produtividade”; Ricerca Finalizzata GR11-172. https://doi.org/10.6084/m9.figshare.c.5713034
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- 2021
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24. The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma
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Manuela Iezzi, Annalisa Pezzolo, Ferdinando Di Cunto, Sara Cabodi, Alessandra Eva, Paola Defilippi, Alessandro Morellato, Silvia Grasso, Alessia Lamolinara, Davide Cangelosi, F. Bianchi, Iris Chiara Salaroglio, Andrea Saglietto, Luigi Varesio, Jennifer Chapelle, Valeria Poli, Chiara Riganti, Marzia Ognibene, Emilia Turco, Costanza Angelini, Federica Fusella, Vincenzo Salemme, Giorgia Centonze, and Melissa Alzona
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business.industry ,Molecular biology ,Signal transducing adaptor protein ,Cell Biology ,medicine.disease ,Pediatric cancer ,Article ,Loss of heterozygosity ,p140Cap ,neuroblastoma ,Paediatric cancer ,Breast cancer ,Neuroblastoma ,medicine ,Cancer research ,Doxorubicin ,business ,Etoposide ,medicine.drug ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13–15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.
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- 2019
25. A Proteomic Analysis of GSD-1a in Mouse Livers: Evidence for Metabolic Reprogramming, Inflammation, and Macrophage Polarization
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Roberta Resaz, Andrea Petretto, Antonio Sica, Irma Colombo, Luigi Varesio, Daniela Segalerba, Alessandra Eva, Davide Cangelosi, Luca Mastracci, Federica Grillo, Federica Raggi, Maria Carla Bosco, and Marzia Ognibene
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Proteomics ,0301 basic medicine ,macrophage polarization ,animal model ,glycogen storage disease type 1a ,hepatocellular adenoma ,hypoxia ,proteomics ,Macrophage polarization ,Inflammation ,Glycogen Storage Disease Type I ,Biology ,Biochemistry ,Mice ,03 medical and health sciences ,Liver disease ,Downregulation and upregulation ,Tandem Mass Spectrometry ,medicine ,Animals ,Glycogen storage disease ,Mice, Knockout ,030102 biochemistry & molecular biology ,Macrophages ,Wild type ,Proteins ,General Chemistry ,Hepatocellular adenoma ,medicine.disease ,Molecular biology ,Disease Models, Animal ,030104 developmental biology ,Liver ,Glucose-6-Phosphatase ,medicine.symptom ,Chromatography, Liquid - Abstract
Glycogen storage disease type 1a (GSD-1a) is a rare genetic disease caused by mutations in the catalytic subunit of the enzyme glucose-6-phosphatase-alpha (G6Pase-α). The majority of patients develop long-term complications including renal failure and hepatocellular adenoma/carcinoma. The purpose of this study was to ascertain the proteomic changes in the liver of LS- G6pc-/- mice, a murine model of GSD-1a, in comparison with wild type mice to identify potential biomarkers of the pathophysiology of the affected liver. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze liver lysates from a total of 20 LS- G6pc-/- and 18 wild type (WT) mice. We compared the proteomic expression profile of LS- G6pc-/- and WT mice. We identified 4138 significantly expressed proteins, 1243 of which were differentially represented. Network and pathway analyses indicate that LS- G6pc-/- livers display an age-dependent modulation of the expression of proteins involved in specific biological processes associated with increased progression of liver disease. Moreover, we found upregulation of proteins involved in the process of tissue inflammation and macrophage polarization toward the M2 phenotype in LS- G6pc-/- mice with adenomas. Our results identify a metabolic reprogramming of glucose-6-P and a pathologic environment in the liver compatible with tumor development and progression.
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- 2019
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26. Abstract 1276: Nucleolin has prognostic value in neuroblastoma patients
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Davide Cangelosi, Chiara Brignole, Veronica Bensa, Roberto Tamma, Fabiana Malaguti, Barbara Carlini, Enzo Calarco, Patrizia Perri, Domenico Ribatti, Nuno A. Fonseca, Joao N. Moreira, Alessandra Eva, Loredana Amoroso, Massimo Conte, Alberto Garaventa, Angela R. Sementa, Maria V. Corrias, Mirco Ponzoni, and Fabio Pastorino
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Cancer Research ,Oncology - Abstract
Background: Neuroblastoma (NB) represents the most frequent form of extracranial solid tumor of infants, responsible for 15% of childhood cancer deaths. Nucleolin (NCL) prognostic value in NB was here investigated. Methods: NCL protein expression was evaluated in NB patients at diagnosis and after induction chemotherapy. NCL mRNA prognostic value was first assessed in a cohort of 20 stage M NB patients and confirmed in the MultiPlatform786 including 786 NB patients of all stages. Overall and event-free survival curves were plotted by Kaplan-Meier method and compared by log-rank test. Findings: NCL protein resulted significantly overexpressed in NB tumors compared to the non tumoral counterpart, and higher in stage M compared to stage L patients. In the stage M cohort and in MultiPlatform786 dataset, patients with high NCL mRNA expression revealed a significant lower survival probability than those with low NCL expression. In MultiPlatform786 dataset, NCL mRNA expression was significantly higher in patients with age >18 months, in stage M and in MYCN amplified tumors than in patients with age ˂18 months and in stage L or MS and with MYCN non amplified tumors, respectively. Multivariate analysis suggested NCL has a significant prognostic value even in the model adjusted for established prognostic markers. NCL significantly stratified patients with age 18, stage M, >18 months and stage M tumor, stages L or MS, and with MYCN not amplified. A significant correlation between NCL and MYCN, MYC, and TERT was found in two independent datasets. Gene set enrichment analysis revealed a significant positive enrichment of MYC target genes and genes involved in telomerase maintenance. NCL protein resulted down-modulated after chemotherapy, in association with morphological features of neuroblastic differentiation. Interpretation: NCL is a novel and independent prognostic marker for NB. Funding: IMH-EuroNanoMed II-2015 (ER-2015-2360441-Eranet) and AIRC IG n. 24397 to PF. Citation Format: Davide Cangelosi, Chiara Brignole, Veronica Bensa, Roberto Tamma, Fabiana Malaguti, Barbara Carlini, Enzo Calarco, Patrizia Perri, Domenico Ribatti, Nuno A. Fonseca, Joao N. Moreira, Alessandra Eva, Loredana Amoroso, Massimo Conte, Alberto Garaventa, Angela R. Sementa, Maria V. Corrias, Mirco Ponzoni, Fabio Pastorino. Nucleolin has prognostic value in neuroblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1276.
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- 2022
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27. MO036DAPAGLIFLOZIN RESCUES THE RENAL PHENOTYPE OF GLYCOGEN STORAGE DISEASE TYPE IB
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Giovambattista Capasso, Francesco Trepiccione, Anna Iervolino, Sabrina Siccardi, Roberta Resaz, Donato Sardella, Mariavittoria D'Acierno, and Alessandra Eva
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Transplantation ,medicine.medical_specialty ,Endocrinology ,Nephrology ,business.industry ,Internal medicine ,Glycogen Storage Disease Type Ib ,medicine ,business ,Phenotype - Abstract
Background and Aims Glycogenosis I type b (GsdI-b) is a rare metabolic disease and immune disorder characterized by hepato-renal glycogen accumulation caused by a deficiency in the Glucose-6-phosphate transporter (G6PT). G6PT transports glucose-6-phosphate (G6P) from cytoplasm to endoplasmic reticulum (ER) where a G6Pase catalyses the hydrolysis of G6P in glucose and phosphate. G6PT deficiency lead to impaired glucose homeostasis, myeloid disfunction and long-term risk of hepatocellular adenomas. No causal therapy is so far available for GSDI-b patients besides a dietary approach to control glycemia and the use of Granulocyte Colony-Stimulating Factor (GCSF) to improve neutropenia. Over time, these supports increase the chronicity of GSDI-b with some complications. A mouse model recapitulating the GDSI-b has been recently generated by inducing G6PT suppression after tamoxifen injection. Here, we characterized the renal phenotype of TM-G6PT-/- mice model focusing on the molecular mechanisms that lead to renal dysfunction. Finally, we evaluated the efficiency of Dapagliflozin, a selective inhibitor of SGLT2, on kidney functions in terms of therapeutic effect. Method Machine learning approach to computer based evaluation of renal morphology was used to analyze the renal sections from TM-G6PT-/- treated with or without dapagliflozin. Results: G6PT is expressed in all renal zones and a severe downregulation of G6PT mRNA expression in whole kidney of TM-G6PT-/- mice can be observed. TM-G6PT-/- mice show tubular vacuolization and overall cellular dysfunction of PT due to a high glycogen accumulation. TM-G6PT-/- mice manifest glycosuria, phosphaturia and polyuria associated with a down regulation of main transporters of PT cells. The urine concentrating defect is due to a primarily role of G6PT in CNT/CD cells confirmed by a downregulation of AQP2, main water channel along CD segments. This mouse model recapitulates the human GSD-Ib renal phenotype characterized by a disfunction of PT but also CNT/CD cells. In order to evaluate whether targeting the glucose metabolism would improve the renal phenotype of these mice we limited glucose flux across the apical membrane of PT cells, applying the SGLT2-inhibitor dapagliflozin to reduce new glycogen formation. After one month of treatment, Dapagliflozin prevents glycogen accumulation in TM-G6PT-/- mice and ameliorates the main dysregulated markers of PT function. This finding was paralleled by an improvement of the histological features of kidney morphology in dapagliflozin treated TM-G6PT-/- mice. Conclusion Our data provide evidence that treatment with dapagliflozin ameliorates intracellular glycogen storage and improves the renal functions in TM-G6PT-/- mice.
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- 2021
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28. The SGLT2-inhibitor dapagliflozin improves neutropenia and neutrophil dysfunction in a mouse model of the inherited metabolic disorder GSDIb
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Alessandra Eva, Paola Marcolongo, Roberta Resaz, Chiara Lavarello, Simonetta Astigiano, Maria Veiga-da-Cunha, Daniela Melis, Federica Raggi, Antonia Assunto, Andrea Petretto, Alessandra Gamberucci, Maria Carla Bosco, Daniela Segalerba, Francesco Trepiccione, Mariavittoria D'Acierno, Resaz, R., Raggi, F., Segalerba, D., Lavarello, C., Gamberucci, A., Bosco, M. C., Astigiano, S., Assunto, A., Melis, D., D'Acierno, M., Veiga-da-Cunha, M., Petretto, A., Marcolongo, P., Trepiccione, F., and Eva, A.
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G-CSF, granulocyte colony stimulating factor ,Medicine (General) ,Neutrophils ,1,5AG, 1,5-anhydroglucitol ,Pharmacology ,Inflammatory bowel disease ,chemistry.chemical_compound ,CFU, colony forming units ,Endocrinology ,1,5-anhydroglucitol-6-phosphate ,5-anhydroglucitol-6-phosphate ,NET, neutrophil extracellular trap ,PRM, parallel reaction monitoring ,GSDIb, Glycogen Storage Disease type 1b ,Medicine ,Glycogen storage disease ,Dapagliflozin ,Biology (General) ,1,5AG6P, 1,5-anhydroglucitol-6-phosphate ,M-CSF, macrophage colony stimulating factor ,Metabolic disorder ,Neutrophil ,Genetic disorder ,Glucose analog ,PMA, phorbol myristate acetate ,Renal sodium-glucose co-transporter-2 ,SGLT2 Inhibitor ,Glycogen storage disease type 1b ,QH301-705.5 ,Short Communication ,Neutropenia ,Mouse model ,R5-920 ,b15-anhydroglucitol-6-phosphate ,Genetics ,G6PC3, glucose-6-phosphatase C3 ,Molecular Biology ,TM, tamoxifen ,business.industry ,medicine.disease ,fMLP, N-formyl-L-methionyl-L-leucyl-phenylalanine ,SGLT2, sodium-glucose co-transporter-2 ,chemistry ,Glycogen storage disease type 1 ,BM, bone marrow ,G6PT, glucose-6-phospate translocase ,business - Abstract
Glycogen Storage Disease type 1b (GSDIb) is a genetic disorder with long term severe complications. Accumulation of the glucose analog 1,5-anhydroglucitol-6-phosphate (1,5AG6P) in neutrophils inhibits the phosphorylation of glucose in these cells, causing neutropenia and neutrophil dysfunctions. This condition leads to serious infections and inflammatory bowel disease (IBD) in GSDIb patients. We show here that dapagliflozin, an inhibitor of the renal sodium-glucose co-transporter-2 (SGLT2), improves neutrophil function in an inducible mouse model of GSDIb by reducing 1,5AG6P accumulation in myeloid cells.
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- 2021
29. Hypoxia Predicts Poor Prognosis in Neuroblastoma Patients and Associates with Biological Mechanisms Involved in Telomerase Activation and Tumor Microenvironment Reprogramming
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Maria Carla Bosco, Nicolò Zanardi, Ulrich Pfeffer, Marco Muselli, Angela Rita Sementa, Martina Morini, Massimo Conte, Luigi Varesio, Davide Cangelosi, Alessandra Eva, and Alberto Garaventa
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0301 basic medicine ,Cancer Research ,Telomerase ,lcsh:RC254-282 ,Article ,Pathogenesis ,03 medical and health sciences ,neuroblastoma ,0302 clinical medicine ,cell immortalization ,Neuroblastoma ,Gene expression ,Medicine ,Tumor microenvironment ,business.industry ,hypoxia ,therapeutic target ,Hypoxia (medical) ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,medicine.disease ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Cancer research ,Biomarker (medicine) ,prognosis ,medicine.symptom ,business ,Reprogramming - Abstract
The biological and clinical heterogeneity of neuroblastoma (NB) demands novel biomarkers and therapeutic targets in order to drive the most appropriate treatment for each patient. Hypoxia is a condition of low-oxygen tension occurring in poorly vascularized tumor tissues. In this study, we aimed to assess the role of hypoxia in the pathogenesis of NB and at developing a new clinically relevant hypoxia-based predictor of outcome. We analyzed the gene expression profiles of 1882 untreated NB primary tumors collected at diagnosis and belonging to four existing data sets. Analyses took advantage of machine learning methods. We identified NB-hop, a seven-gene hypoxia biomarker, as a predictor of NB patient prognosis, which is able to discriminate between two populations of patients with unfavorable or favorable outcome on a molecular basis. NB-hop retained its prognostic value in a multivariate model adjusted for established risk factors and was able to additionally stratify clinically relevant groups of patients. Tumors with an unfavorable NB-hop expression showed a significant association with telomerase activation and a hypoxic, immunosuppressive, poorly differentiated, and apoptosis-resistant tumor microenvironment. NB-hop defines a new population of NB patients with hypoxic tumors and unfavorable prognosis and it represents a critical factor for the stratification and treatment of NB patients.
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- 2020
30. Transcriptome analysis defines myocardium gene signatures in children with ToF and ASD and reveals disease-specific molecular reprogramming in response to surgery with cardiopulmonary bypass
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Massimo Acquaviva, Martina Morini, Davide Cangelosi, Giuseppe Panizzon, Maria Luisa Belli, Maria Carla Bosco, Pamela Becherini, Alessandra Eva, Giuseppe Cervo, Federica Raggi, Fabiola Blengio, and Luigi Varesio
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Therapeutic gene modulation ,lcsh:Medicine ,Bioinformatics ,Heart Septal Defects, Atrial ,General Biochemistry, Genetics and Molecular Biology ,Atrial septal defects ,Transcriptome ,Pathogenesis ,Gene expression ,Transcriptional regulation ,Medicine ,Humans ,Hypoxia ,Child ,Gene ,Congenital heart disease ,Cardiopulmonary Bypass ,business.industry ,Atrial ,Research ,Gene Expression Profiling ,Heart Septal Defects ,Myocardium ,lcsh:R ,General Medicine ,Atrial myocardium ,Cardiopulmonary bypass ,Gene expression profiling ,Tetralogy of Fallot ,business - Abstract
Background Tetralogy of Fallot (ToF) and Atrial Septal Defects (ASD) are the most common types of congenital heart diseases and a major cause of childhood morbidity and mortality. Cardiopulmonary bypass (CPB) is used during corrective cardiac surgery to support circulation and heart stabilization. However, this procedure triggers systemic inflammatory and stress response and consequent increased risk of postoperative complications. The aim of this study was to define the molecular bases of ToF and ASD pathogenesis and response to CPB and identify new potential biomarkers. Methods Comparative transcriptome analysis of right atrium specimens collected from 10 ToF and 10 ASD patients was conducted before (Pre-CPB) and after (Post-CPB) corrective surgery. Total RNA isolated from each sample was individually hybridized on Affymetrix HG-U133 Plus Array Strips containing 38,500 unique human genes. Differences in the gene expression profiles and functional enrichment/network analyses were assessed using bioinformatic tools. qRT-PCR analysis was used to validate gene modulation. Results Pre-CPB samples showed significant differential expression of a total of 72 genes, 28 of which were overexpressed in ToF and 44 in ASD. According to Gene Ontology annotation, the mostly enriched biological processes were represented by matrix organization and cell adhesion in ToF and by muscle development and contractility in ASD specimens. GSEA highlighted the specific enrichment of hypoxia gene sets in ToF samples, pointing to a role for hypoxia in disease pathogenesis. The post-CPB myocardium exhibited significant alterations in the expression profile of genes related to transcription regulation, growth/apoptosis, inflammation, adhesion/matrix organization, and oxidative stress. Among them, only 70 were common to the two disease groups, whereas 110 and 24 were unique in ToF and ASD, respectively. Multiple functional interactions among differentially expressed gene products were predicted by network analysis. Interestingly, gene expression changes in ASD samples followed a consensus hypoxia profile. Conclusion Our results provide a comprehensive view of gene reprogramming in right atrium tissues of ToF and ASD patients before and after CPB, defining specific molecular pathways underlying disease pathophysiology and myocardium response to CPB. These findings have potential translational value because they identify new candidate prognostic markers and targets for tailored cardioprotective post-surgical therapies.
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- 2020
31. MYCN Regulates Metabolism Through Vesicular Transfer of Glycolytic Kinases
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Martin R. Larsen, Victor Corasolla Carregari, Alessandra Eva, Alexia Tsakaneli, Olesya Chayka, Evon Poon, Arturo Sala, Louis Chesler, Luisa Pieroni, Giuseppe Palmisano, Martina Morini, Evgeni Makarov, and Giuliana Cangemi
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Kinase ,Chemistry ,Neuroblastoma ,medicine ,Cancer ,Glycolysis ,Metabolism ,PKM2 ,medicine.disease ,neoplasms ,Warburg effect ,Pyruvate kinase ,Cell biology - Abstract
Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer promoting activity of extracellular vesicles to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYCN expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and Hexokinase II were detected in the EVs circulating in the blood stream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC activated cancers might spread oncogenic signals to remote body locations through extracellular vesicles.
- Published
- 2020
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32. Circulating exosomal microRNA as potential biomarkers of hepatic injury and inflammation inGlycogen storage disease type 1a
- Author
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Davide Cangelosi, Martina Morini, Federica Grillo, Luca Mastracci, Cristina Bottino, Irma Colombo, Alessandra Eva, Roberta Resaz, Daniela Segalerba, and Maria Carla Bosco
- Subjects
0301 basic medicine ,Chemokine ,Adenoma ,biology ,business.industry ,Metabolic disorder ,Neuroscience (miscellaneous) ,Medicine (miscellaneous) ,Inflammation ,Hepatocellular adenoma ,medicine.disease ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Immunology and Microbiology (miscellaneous) ,030220 oncology & carcinogenesis ,Hepatocellular carcinoma ,microRNA ,medicine ,Cancer research ,biology.protein ,Glycogen storage disease ,medicine.symptom ,business - Abstract
Most patients affected by Glycogen storage disease type 1a (GSD-1a), an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-alpha (G6Pase-α), develop renal and liver complications, including development of hepatocellular adenoma/carcinoma. The purpose of this study was to identify potential biomarkers of the pathophysiology of the GSD1a affected liver. To this end, we utilized the plasma exosomes of a murine model of GSD-1a, LS-G6pc−/− mice, to uncover microRNA expression modulation associated with the disease. Differentially expressed microRNA between LS-G6pc−/− and wild type mice, LS-G6pc−/− mice with hepatocellular adenoma and LS-G6pc−/− mice without adenoma, and LS-G6pc−/− mice with amyloidosis and LS-G6pc−/− mice without amyloidosis were identified. Pathway analysis demonstrated that the target genes of the differentially expressed microRNA were significantly enriched for insulin signaling pathway, glucose and lipid metabolism, Wnt-beta catenin, telomere maintenance and hepatocellular carcinoma, and chemokine and immune regulation signaling pathways. While some microRNA were common to the different pathologic conditions others were unique to cancerous or inflammatory status of the animals. Therefore, the altered expression of several microRNA correlates with various pathologic liver statuses and may help discriminate during the progression of the disease and the development of late GSD1-a associated complications.
- Published
- 2020
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33. CHL1 gene acts as a tumor suppressor in human neuroblastoma
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Gabriella Pagnan, Davide Cangelosi, Danilo Marimpietri, Luigi Varesio, Renata Boldrini, Francesco Frassoni, Maria Chiara Benedetti, Alessandra Eva, Marzia Ognibene, Michele Cilli, Annalisa Pezzolo, Vito Pistoia, and Alberto Garaventa
- Subjects
0301 basic medicine ,autophagy ,Neurite ,Cell growth ,apoptosis ,differentiation ,Biology ,medicine.disease ,Pediatric cancer ,CHL1 ,neuroblastoma ,03 medical and health sciences ,030104 developmental biology ,Oncology ,Tumor progression ,Neuroblastoma ,Cancer research ,medicine ,CHL1 gene ,Neuronal Cell Adhesion Molecule ,Protein kinase B ,Research Paper - Abstract
Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.
- Published
- 2018
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34. Connectivity Map Analysis Indicates PI3K/Akt/mTOR Inhibitors as Potential Anti-Hypoxia Drugs in Neuroblastoma
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Alessandra Eva, Paolo Uva, Davide Cangelosi, Maria Carla Bosco, Loredana Amoroso, Massimo Conte, and Alberto Garaventa
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0301 basic medicine ,Cancer Research ,Biology ,Article ,neuroblastoma ,03 medical and health sciences ,0302 clinical medicine ,Neuroblastoma ,Gene expression ,medicine ,Gene ,Protein kinase B ,RC254-282 ,PI3K/AKT/mTOR pathway ,treatment ,hypoxia ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Hypoxia (medical) ,medicine.disease ,Drug repositioning ,030104 developmental biology ,Oncology ,Cell culture ,030220 oncology & carcinogenesis ,PI3K/Akt/mTOR inhibitors ,Cancer research ,medicine.symptom - Abstract
Simple Summary A large percentage of patients with neuroblastoma relapse and die, despite treatment, thus demanding new personalized strategies and therapeutic targets. Hypoxia, a condition of reduced oxygenation in several solid tumors, has profound effects on the neuroblastoma (NB) tumor biology and patient prognosis. Establishing new connections between hypoxia and pharmacological compounds may provide novel treatment strategies for NB patients. In the present study, we successfully identified 19 compounds mainly belonging to the class of PI3K/Akt/mTOR inhibitors, whose anti-hypoxia effect was shown on the gene expression profile of nine distinct cell lines using connectivity map software. We independently confirmed these findings on NB cells cultured under hypoxia conditions and treated with the mTORC inhibitor PP242. PI3K/Akt/mTOR inhibitors represent a potential effective class of compounds targeting hypoxia in neuroblastoma. PI3K/Akt/mTOR inhibitors may thus find future applicability as a new adjuvant therapy in randomized clinical trials involving neuroblastoma patients with hypoxic tumors. Abstract Neuroblastoma (NB) is one of the deadliest pediatric cancers, accounting for 15% of deaths in childhood. Hypoxia is a condition of low oxygen tension occurring in solid tumors and has an unfavorable prognostic factor for NB. In the present study, we aimed to identify novel promising drugs for NB treatment. Connectivity Map (CMap), an online resource for drug repurposing, was used to identify connections between hypoxia-modulated genes in NB tumors and compounds. Two sets of 34 and 21 genes up- and down-regulated between hypoxic and normoxic primary NB tumors, respectively, were analyzed with CMap. The analysis reported a significant negative connectivity score across nine cell lines for 19 compounds mainly belonging to the class of PI3K/Akt/mTOR inhibitors. The gene expression profiles of NB cells cultured under hypoxic conditions and treated with the mTORC complex inhibitor PP242, referred to as the Mohlin dataset, was used to validate the CMap findings. A heat map representation of hypoxia-modulated genes in the Mohlin dataset and the gene set enrichment analysis (GSEA) showed an opposite regulation of these genes in the set of NB cells treated with the mTORC inhibitor PP242. In conclusion, our analysis identified inhibitors of the PI3K/Akt/mTOR signaling pathway as novel candidate compounds to treat NB patients with hypoxic tumors and a poor prognosis.
- Published
- 2021
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35. Secondary Somatic Mutations in G-Protein-Related Pathways and Mutation Signatures in Uveal Melanoma
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D. A. Coviello, Ulrich Pfeffer, Alessandra Eva, Cinzia Bernardi, Francesca Piaggio, Roberto Puzone, Adriana Amaro, Michela Croce, Michael Zeschnigk, Silvia Viaggi, Annalisa Barla, Davide Cangelosi, Pieter A. van der Velden, Veronica Tozzo, Martine J. Jager, and Serena Patrone
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0301 basic medicine ,Cancer Research ,tumor evolution ,Medizin ,Biology ,medicine.disease_cause ,lcsh:RC254-282 ,Article ,driver mutation ,gene set enrichment ,mutation signature ,03 medical and health sciences ,0302 clinical medicine ,medicine ,KEGG ,Gene ,Genetics ,Mutation ,BAP1 ,PTK2B ,GNA11 ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,030104 developmental biology ,Cysteinyl leukotriene receptor 2 ,Oncology ,030220 oncology & carcinogenesis ,GNAQ - Abstract
Background: Uveal melanoma (UM), a rare cancer of the eye, is characterized by initiating mutations in the genes G-protein subunit alpha Q (GNAQ), G-protein subunit alpha 11 (GNA11), cysteinyl leukotriene receptor 2 (CYSLTR2), and phospholipase C beta 4 (PLCB4) and by metastasis-promoting mutations in the genes splicing factor 3B1 (SF3B1), serine and arginine rich splicing factor 2 (SRSF2), and BRCA1-associated protein 1 (BAP1). Here, we tested the hypothesis that additional mutations, though occurring in only a few cases (&ldquo, secondary drivers&rdquo, ), might influence tumor development. Methods: We analyzed all the 4125 mutations detected in exome sequencing datasets, comprising a total of 139 Ums, and tested the enrichment of secondary drivers in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that also contained the initiating mutations. We searched for additional mutations in the putative secondary driver gene protein tyrosine kinase 2 beta (PTK2B) and we developed new mutational signatures that explain the mutational pattern observed in UM. Results: Secondary drivers were significantly enriched in KEGG pathways that also contained GNAQ and GNA11, such as the calcium-signaling pathway. Many of the secondary drivers were known cancer driver genes and were strongly associated with metastasis and survival. We identified additional mutations in PTK2B. Sparse dictionary learning allowed for the identification of mutational signatures specific for UM. Conclusions: A considerable part of rare mutations that occur in addition to known driver mutations are likely to affect tumor development and progression.
- Published
- 2019
36. PIPE-T: a new Galaxy tool for the analysis of RT-qPCR expression data
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Maria Carla Bosco, Martina Morini, Luigi Varesio, Marco Antonio Tangaro, Federico Zambelli, Nicolò Zanardi, Alessandra Eva, and Davide Cangelosi
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0301 basic medicine ,Source code ,Statistical methods ,Computer science ,media_common.quotation_subject ,education ,lcsh:Medicine ,Gene Expression ,Real-Time Polymerase Chain Reaction ,computer.software_genre ,Article ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,Software ,law ,Gene expression ,lcsh:Science ,Transcriptomics ,Polymerase chain reaction ,media_common ,Measure (data warehouse) ,Multidisciplinary ,business.industry ,cloud computing ,lcsh:R ,Computational Biology ,Genomics ,pipe-t ,Laniakea ,Galaxy ,Reverse transcriptase ,030104 developmental biology ,Expression data ,Data Interpretation, Statistical ,lcsh:Q ,Data mining ,Transcriptome ,business ,computer ,030217 neurology & neurosurgery - Abstract
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and fast method to measure gene expression. Reproducibility of the analyses is the main limitation of RT-qPCR experiments. Galaxy is an open, web-based, genomic workbench for a reproducible, transparent, and accessible science. Our aim was developing a new Galaxy tool for the analysis of RT-qPCR expression data. Our tool was developed using Galaxy workbench version 19.01 and functions implemented in several R packages. We developed PIPE-T, a new Galaxy tool implementing a workflow, which offers several options for parsing, filtering, normalizing, imputing, and analyzing RT-qPCR data. PIPE-T requires two input files and returns seven output files. We tested the ability of PIPE-T to analyze RT-qPCR data on two example datasets available in the gene expression omnibus repository. In both cases, our tool successfully completed execution returning expected results. PIPE-T can be easily installed from the Galaxy main tool shed or from Docker. Source code, step-by-step instructions, and example files are available on GitHub to assist new users to install, execute, and test PIPE-T. PIPE-T is a new tool suitable for the reproducible, transparent, and accessible analysis of RT-qPCR expression data.
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- 2019
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37. Correction to: The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma
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Alessia Lamolinara, Sara Cabodi, Alessandra Eva, Manuela Iezzi, Annalisa Pezzolo, Alessandro Morellato, Costanza Angelini, Luigi Varesio, Jennifer Chapelle, Federica Fusella, Davide Cangelosi, Andrea Saglietto, Valeria Poli, Silvia Grasso, Iris Chiara Salaroglio, Giorgia Centonze, Vincenzo Salemme, F. Bianchi, Paola Defilippi, Emilia Turco, Chiara Riganti, Marzia Ognibene, Melissa Alzona, and Ferdinando Di Cunto
- Subjects
Male ,Lung Neoplasms ,Cell Survival ,Mice, SCID ,Mice ,Neuroblastoma ,Mice, Inbred NOD ,Biomarkers, Tumor ,Tumor Cells, Cultured ,Medicine ,Animals ,Humans ,RNA, Messenger ,Molecular Biology ,Cell Proliferation ,business.industry ,Signal transducing adaptor protein ,Correction ,Infant ,Cell Biology ,Neoplasms, Experimental ,medicine.disease ,Cell biology ,Adaptor Proteins, Vesicular Transport ,business - Abstract
Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13-15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.
- Published
- 2019
38. SP014RENAL PHENOTYPE OF A MOUSE MODEL OF GLYCOGENOSIS I TYPE B
- Author
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Luigi R De La Motte, Alessandra Eva, Francesco Trepiccione, Giovambattista Capasso, Roberta Resaz, Mariavittoria D'Acierno, Anna Iervolino, Yoko Suzumoto, and Sabrina Siccardi
- Subjects
Genetics ,Transplantation ,Nephrology ,business.industry ,Medicine ,business ,Phenotype - Published
- 2019
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39. Characterization of high- and low-risk hepatocellular adenomas by magnetic resonance imaging in an animal model of glycogen storage disease type 1A
- Author
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Federica Grillo, Roberta Resaz, Francesca La Rosa, Luca Basso, Maria Carla Bosco, Carlo Emanuele Neumaier, Andrea Puglisi, Alessandra Eva, Daniela Segalerba, Luigi Varesio, and Luca Mastracci
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Contrast enhancement ,β-Catenin ,Neuroscience (miscellaneous) ,Medicine (miscellaneous) ,lcsh:Medicine ,Glycogen Storage Disease Type I ,General Biochemistry, Genetics and Molecular Biology ,Malignant transformation ,Adenoma, Liver Cell ,03 medical and health sciences ,0302 clinical medicine ,Animal model ,Immunology and Microbiology (miscellaneous) ,medicine ,lcsh:Pathology ,Glycogen storage disease ,Animals ,Liver ,Magnetic resonance ,Tumor ,medicine.diagnostic_test ,business.industry ,Liver Neoplasms ,lcsh:R ,Magnetic resonance imaging ,medicine.disease ,Magnetic Resonance Imaging ,Patient management ,Disease Models, Animal ,030104 developmental biology ,Organ Specificity ,Hepatocellular carcinoma ,Glucose-6-Phosphatase ,Immunohistochemistry ,business ,030217 neurology & neurosurgery ,Research Article ,lcsh:RB1-214 - Abstract
Hepatocellular adenomas (HCAs) are benign tumors, of which the most serious complications are hemorrhage and malignant transformation to hepatocellular carcinoma (HCC). Among the various subtypes of HCA, the β-catenin-activated subtype (bHCA) is associated with greatest risk of malignant transformation. Magnetic resonance imaging (MRI) is an important tool to differentiate benign and malignant hepatic lesions, and preclinical experimental approaches may help to develop a method to identify MRI features associated with bHCA. HCAs are associated with various pathologies, including glycogen storage disease 1a (GSD1a). Here, we utilized a mouse model for GSD1a that develops HCA and HCC, and analyzed the mice in order to distinguish low-risk from high-risk tumors. Animals were scanned by MRI using a hepato-specific contrast agent. The mice were sacrificed after MRI and their lesions were classified using immunohistochemistry. We observed that 45% of the animals developed focal lesions, and MRI identified four different patterns after contrast administration: isointense, hyperintense and hypointense lesions, and lesions with peripheral contrast enhancement. After contrast administration, only bHCA and HCC were hypointense in T1-weighted imaging and mildly hyperintense in T2-weighted imaging. Thus, high-risk adenomas display MRI features clearly distinguishable from those exhibited by low-risk adenomas, indicating that MRI is a reliable method for early diagnosis and classification of HCA, necessary for correct patient management., Summary: High-risk adenomas display magnetic resonance imaging (MRI) features clearly distinguishable from those exhibited by low-risk adenomas, suggesting the possibility of applying MRI to humans for non-invasive diagnostic purposes.
- Published
- 2019
40. How many mutations does it take to make a uveal melanoma?
- Author
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Piaggio, Francesca, Tozzo, Veronica, Bernardi, Cinzia, Michela, Croce, LO SICCO, Claudia, Gangemi, Rosaria, Roberto, Puzone, Viaggi, Silvia, Patrone, Serena, Barla, Annalisa, Coviello, Domenico, Jager, Martine J., van der Velden, Pieter A., Davide, Cangelosi, Alessandra, Eva, Ferrini, Silvano, Pfeffer, Ulrich, and Amaro, ADRIANA AGNESE
- Published
- 2019
41. MCM2 and Carbonic Anhydrase 9 Are Novel Potential Targets for Neuroblastoma Pharmacological Treatment
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Delfina Costa, Tullio Florio, Alessandra Eva, Aldo Pagano, Gian Paolo Tonini, Stefano Thellung, Davide Cangelosi, Francesca Baldini, Carlo Zanon, Matilde Calderoni, Patrizia Garbati, Raffaella Barbieri, and Paola Modesto
- Subjects
Cisplatin ,carbonic anhydrase 9 ,Chemistry ,Cellular differentiation ,Medicine (miscellaneous) ,Carbonic Anhydrase 9 ,medicine.disease ,Article ,General Biochemistry, Genetics and Molecular Biology ,In vitro ,neuroblastoma ,acetazolamide ,MCM2 ,lcsh:Biology (General) ,ciprofloxacin ,In vivo ,Neuroblastoma ,Gene expression ,Cancer research ,medicine ,Acetazolamide ,lcsh:QH301-705.5 ,medicine.drug - Abstract
To overcome the lack of effective pharmacological treatments for high-risk neuroblastoma (HR-NB), the development of novel in vitro and in vivo models that better recapitulate the disease is required. Here, we used an in vitro multiclonal cell model encompassing NB cell differentiation stages, to identify potential novel pharmacological targets. This model allowed us to identify, by low-density RT-PCR arrays, two gene sets, one over-expressed during NB cell differentiation, and the other up-regulated in more malignant cells. Challenging two HR-NB gene expression datasets, we found that these two gene sets are related to high and low survival, respectively. Using mouse NB cisplatin-treated xenografts, we identified two genes within the list associated to the malignant stage (MCM2 and carbonic anhydrase 9), whose expression is positively correlated with tumor growth. Thus, we tested their pharmacological targeting as potential therapeutic strategy. We measured mice survival and tumor growth rate after xenografts of human NB treated with cisplatin in the presence of MCM2/carbonic anhydrase 9 inhibitors (ciprofloxacin and acetazolamide). MCM2 or carbonic anhydrase 9 inhibition significantly increased cisplatin activity, supporting their possible testing for NB therapy.
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- 2020
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42. Western blot analysis v4
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Marzia Ognibene, Davide Cangelosi, Martina Morini, Daniela Segalerba, Maria Carla Bosco, Angela Rita Sementa, Alessandra Eva, and Luigi Varesio
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Western blot ,medicine.diagnostic_test ,medicine ,Biology ,Molecular biology - Published
- 2017
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43. Immunocytochemical and immunohistochemical analysis v4
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Marzia Ognibene, Davide Cangelosi, Martina Morini, Daniela Segalerba, Maria Carla Bosco, Angela Rita Sementa, Alessandra Eva, and Luigi Varesio
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Pathology ,medicine.medical_specialty ,medicine - Published
- 2017
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44. Regulation of Human Macrophage M1-M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1
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Luigi Varesio, Marco Gattorno, Federica Raggi, Federica Penco, Mirella Giovarelli, Daniele Pierobon, Francesco Novelli, Simone Pelassa, Alessandra Eva, and Maria Carla Bosco
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lcsh:Immunologic diseases. Allergy ,0301 basic medicine ,Chemokine ,T cell ,Immunology ,Inflammation ,Proinflammatory cytokine ,03 medical and health sciences ,medicine ,Immunology and Allergy ,Receptor ,Original Research ,polarization ,immunoregulatory receptors ,biology ,hypoxia ,Hypoxia (medical) ,Phenotype ,Cell biology ,macrophages ,macrophages, hypoxia, polarization, immunoregulatory receptors, inflammation ,030104 developmental biology ,medicine.anatomical_structure ,inflammation ,biology.protein ,medicine.symptom ,lcsh:RC581-607 ,Homing (hematopoietic) - Abstract
Macrophages (Mf) are a heterogeneous population of tissue-resident professional phagocytes and a major component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth. They can undergo diverse forms of activation in response to environmental factors, polarizing into specialized functional subsets. A common hallmark of the pathologic environment is represented by hypoxia. The impact of hypoxia on human Mf polarization has not been fully established. The objective of this study was to elucidate the effects of a hypoxic environment reflecting that occurring in vivo in diseased tissues on the ability of human Mf to polarize into classically activated (proinflammatory M1) and alternatively-activated (anti-inflammatory M2) subsets. We present data showing that hypoxia hinders Mf polarization toward the M1 phenotype by decreasing the expression of T cell costimulatory molecules and chemokine homing receptors and the production of proinflammatory, Th1-priming cytokines typical of classical activation, while promoting their acquisition of phenotypic and secretory features of alternative activation. Furthermore, we identify the triggering receptor expressed on myeloid cells (TREM)-1, a member of the Ig-like immunoregulatory receptor family, as a hypoxia-inducible gene in Mf and demonstrate that its engagement by an agonist Ab reverses the M2-polarizing effect of hypoxia imparting a M1-skewed phenotype to Mf. Finally, we provide evidence that Mf infiltrating the inflamed hypoxic joints of children affected by Oligoarticular Juvenile Idiopatic Arthritis (OJIA) express high surface levels of TREM-1 associated with predominant M1 polarization and suggest the potential of this molecule in driving M1 proinflammatory reprogramming in the hypoxic synovial environment.
- Published
- 2017
45. Development of hepatocellular adenomas and carcinomas in mice with liver-specific G6Pase-α deficiency
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Maria Carla Bosco, Janice Y. Chou, Federica Grillo, Angela Rita Sementa, Daniela Segalerba, Daniele Murgia, Ottavia Barbieri, Cristina Vanni, Luca Mastracci, Luigi Varesio, Alessandra Eva, and Roberta Resaz
- Subjects
Glucose-6-phosphatase-α ,Adenoma ,Hepatic steatosis ,medicine.medical_specialty ,Carcinoma, Hepatocellular ,Hepatocellular carcinoma ,Neuroscience (miscellaneous) ,lcsh:Medicine ,Medicine (miscellaneous) ,Glucosephosphate Dehydrogenase ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Pathogenesis ,Mice ,Immunology and Microbiology (miscellaneous) ,Internal medicine ,lcsh:Pathology ,medicine ,Animals ,Weaning ,Glycogen storage disease ,Glucose homeostasis ,Animal model ,lcsh:R ,Liver Neoplasms ,Hepatocellular adenoma ,medicine.disease ,Endocrinology ,Steatosis ,Glycogen storage disease type 1a ,lcsh:RB1-214 ,Research Article ,Hepatomegaly - Abstract
Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α), and is characterized by impaired glucose homeostasis and a high risk of developing hepatocellular adenomas (HCAs). A globally G6Pase-α-deficient (G6pc−/−) mouse model that shows pathological features similar to those of humans with GSD-1a has been developed. These mice show a very severe phenotype of disturbed glucose homeostasis and rarely live beyond weaning. We generated liver-specific G6Pase-α-deficient (LS‑G6pc−/−) mice as an alternative animal model for studying the long-term pathophysiology of the liver and the potential treatment strategies, such as cell therapy. LS‑G6pc−/− mice were viable and exhibited normal glucose profiles in the fed state, but showed significantly lower blood glucose levels than their control littermates after 6 hours of fasting. LS‑G6pc−/− mice developed hepatomegaly with glycogen accumulation and hepatic steatosis, and progressive hepatic degeneration. Ninety percent of the mice analyzed developed amyloidosis by 12 months of age. Finally, 25% of the mice sacrificed at age 10–20 months showed the presence of multiple HCAs and in one case late development of hepatocellular carcinoma (HCC). In conclusion, LS‑G6pc−/− mice manifest hepatic symptoms similar to those of human GSD-1a and, therefore, represent a valid model to evaluate long-term liver pathogenesis of GSD-1a.
- Published
- 2014
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46. Bradykinin-induced asthmatic fibroblast/myofibroblast activities via bradykinin B2 receptor and different MAPK pathways
- Author
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Leonardo M. Fabbri, Loredana Petecchia, Valentina Sorbello, Anna M. Longo, Fabio Luigi Massimo Ricciardolo, Peter J. Sterk, Antonino Di Stefano, Pieter S. Hiemstra, Giovanni A. Rossi, Fabrizio Luppi, Alessandra Eva, Federica Sabatini, Cristina Vanni, AII - Amsterdam institute for Infection and Immunity, Pulmonology, Sabatini, F, Luppi, F, Petecchia, L, Di Stefano, A, Longo, A, Eva, A, Vanni, C, Hiemstra, P, Sterk, P, Sorbello, V, Fabbri, L, Rossi, G, and Ricciardolo, F
- Subjects
MAPK/ERK pathway ,Receptor, Bradykinin B2 ,B2 RECEPTOR ,Bradykinin B2 receptor ,Receptor expression ,HUMAN BRONCHIAL FIBROBLASTS ,Bradykinin ,p38-mitogen-activated protein kinases ,alpha-smooth muscle actin ,Extracellular-regulated kinase (1)/(2) ,chemistry.chemical_compound ,Epidermal growth factor ,NITRIC-OXIDE SYNTHESIS ,Humans ,Bradykinin B-2 receptor ,Bradykinin receptor ,Myofibroblasts ,Receptor ,Cells, Cultured ,Cell proliferation ,GENE-EXPRESSION ,HUMAN LUNG FIBROBLASTS ,NECROSIS-FACTOR-ALPHA ,GROWTH-FACTOR-BETA ,IN-VITRO ,WOUND CLOSURE ,SIGNALING PATHWAY ,Pharmacology ,Extracellular-regulated kinase ½ ,Epidermal growth factor receptor ,Chemistry ,Receptor transactivation ,Cell Differentiation ,Fibroblasts ,Molecular biology ,Actins ,Asthma ,ErbB Receptors ,α-smooth muscle actin ,Mitogen-Activated Protein Kinases ,Myofibroblast - Abstract
Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B 2 receptor expression by Western blot analysis; cell proliferation by [ 3 H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B 2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B 2 receptor expression in HBAFb. Bradykinin, via bradykinin B 2 receptor, significantly increased fibroblast proliferation at lower concentration (10 −11 M) and α-SMA expression/polymerization at higher concentration (10 −6 M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B 2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B 2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.
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- 2013
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47. Chronic hypoxia reprograms human immature dendritic cells by inducing a proinflammatory phenotype and TREM-1 expression
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Paola Cappello, Mirella Giovarelli, Miriam Filippi, Tiziana Musso, Alessandra Eva, Maria Carla Bosco, Federica Raggi, Fabiola Blengio, Daniele Pierobon, Francesco Novelli, and Luigi Varesio
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Chemokine ,Chemokine receptor ,Innate immune system ,biology ,Downregulation and upregulation ,Immunology ,biology.protein ,Pattern recognition receptor ,Immunology and Allergy ,Acquired immune system ,Cell biology ,Proinflammatory cytokine ,Homing (hematopoietic) - Abstract
DCs are powerful antigen-presenting cells central in the orchestration of innate and acquired immunity. DC development, migration, and activities are intrinsically linked to the microenvironment. DCs migrate through pathologic tissues before reaching their final destination in the lymph nodes. Hypoxia, a condition of low partial oxygen pressure, is a common feature of many pathologic situations, capable of modifying DC phenotype and functional behavior. We studied human monocyte-derived immature DCs generated under chronic hypoxic conditions (H-iDCs). We demonstrate by gene expression profiling the upregulation of a cluster of genes coding for antigen-presentation, immunoregulatory, and pattern recognition receptors, suggesting a stimulatory role for hypoxia on iDC immunoregulatory functions. In particular, we show that H-iDCs express triggering receptor expressed on myeloid cells(TREM-1), a member of the Ig superfamily of immunoreceptors and an amplifier of inflammation. This effect is reversible because H-iDC reoxygenation results in TREM-1 down-modulation. TREM-1 engagement promotes upregulation of T-cell costimulatory molecules and homing chemokine receptors, typical of mature DCs, and increases the production of proinflammatory, Th1/Th17-priming cytokines/chemokines, resulting in increased T-cell responses. These results suggest that TREM-1 induction by the hypoxic microenvironment represents a mechanism of regulation of Th1-cell trafficking and activation by iDCs differentiated at pathologic sites.
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- 2013
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48. The hypoxic environment reprograms the cytokine/chemokine expression profile of human mature dendritic cells
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Paola Cappello, Daniele Pierobon, Alessandra Eva, Federica Raggi, Mirella Giovarelli, Fabiola Blengio, Luigi Varesio, and Maria Carla Bosco
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Vascular Endothelial Growth Factor A ,Immunology ,Biology ,Monocytes ,CCL5 ,Proinflammatory cytokine ,Cell Movement ,Osteogenesis ,cytokine ,Humans ,Immunology and Allergy ,CXCL10 ,CXCL14 ,Cells, Cultured ,CXCL16 ,Immunity, Cellular ,Neovascularization, Pathologic ,hypoxia ,chemokine ,dendritic cells ,Cell Differentiation ,Dendritic Cells ,Hematology ,Cell Hypoxia ,Cell biology ,CCL20 ,CXCL2 ,Cellular Microenvironment ,Gene Expression Regulation ,Cytokines ,Osteopontin ,Chemokines ,Inflammation Mediators ,Transcriptome ,CCL23 - Abstract
Myeloid dendritic cells (DCs) are professional antigen-presenting cells critical for the orchestration of immunity and maintenance of self-tolerance. DC development and functions are tightly regulated by a complex network of inhibitory and activating signals present in the tissue microenvironment, and dysregulated DC responses may result in amplification of inflammation, loss of tolerance, or establishment of immune escape mechanisms. Generation of mature (m)DCs from monocytic precursors recruited at pathological sites occurs under condition of low partial oxygen pressure (pO(2)). However, the way in which the hypoxic microenvironment modulates the functions of these cells is still not clear. We demonstrate that chronic hypoxia (4 days, 1% O(2)) promotes the onset of a highly proinflammatory gene expression profile in mDCs generated from primary human monocytes, characterized by the modulation of a significant cluster of genes coding for proinflammatory chemokines/cytokines and/or their receptors. Within the chemokine system, strong upregulation of genes encoding proteins chemotactic for neutrophils, such as CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8, and for activated/memory T lymphocytes, monocytes, and immature (i) DCs, e.g. CCL20, CCL3 and CCL5, was observed, concomitant with decreased expression of genes coding for naive/resting T cells chemoattractants, CCL18 and CCL23. Other hypoxia-inducible genes coded for cytokines with a primary role in inflammation and angiogenesis, including osteopontin, vascular endothelial growth factor, and IL-1β. mRNA modulation was paralleled by protein secretion. These results suggest that conditions of reduced O(2) availability reprograms mDCs toward a proinflammatory direction by tuning the cytokine/chemokine repertoire, thus affecting their ability to regulate leukocyte trafficking and activation at pathological sites, with potential implications for the pathogenesis of chronic inflammatory diseases.
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- 2013
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49. Accurate stratification of patients with neuroblastoma by application of the reject option paradigm to outcome prediction
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Alessandra Eva, Davide Cangelosi, Martina Morini, Maria Carla Bosco, Luigi Varesio, and Massimo Conte
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Oncology ,medicine.medical_specialty ,business.industry ,Internal medicine ,Neuroblastoma ,Medicine ,Hypoxia (medical) ,medicine.symptom ,business ,Outcome prediction ,medicine.disease ,Surgery - Abstract
Motivation Neuroblastoma (NB) is the most common solid pediatric tumor, deriving from ganglionic lineage precursors of the sympathetic nervous system. Half of the patients with high-risk NB die despite treatment and more accurate outcome prediction models are needed to direct effective therapies. To limit the therapy's side effects, a highly accurate classification of a limited number of patients is preferable to a broader classification with greater error. We aimed at improving the classifier's accuracy and establishing the criteria for confident selection of classification rules by applying the reject option technique (RO). Methods We evaluated outcome prediction by BFTree, ID3, J48, REPTree, SimpleCART decision-tree algorithms utilizing a 182 patients’ dataset. Fifty percent of the patients were utilized for model selection, and the remaining 50% for independent validation. The risk factors were: NB-hypo, age at diagnosis, stage and MYCN amplification. Accuracy of the classification was measured by Matthew’s Correlation Coefficient and assessed by 2 fold cross validation analysis repeated 1000 times. Trade-off between confidence and accuracy was estimated by the RO technique utilizing the accuracy/rejection plot. Kaplan-Meier estimate and log-rank test assessed overall survival. Results Every decision tree classified the patients' outcome with a confidence >0.6. Only ID3, utilizing all risk factors, stratified stage 4 patients and was chosen for further analysis. Application of RO raised the ID3 accuracy from 69% to 92%. This result was obtained because of accuracy/rejection plot identified a threshold confidence of 0.66 at which 71% of the patients, classified by highly reliable rules, were accepted. The trade-off was the exclusion from classification of 29% of patients falling in low represented and ambiguous rules. Kaplan-Meier curves showed a significant stratification of stage 4 patients following RO application. In conclusion, we demonstrated that application of the RO improves the classification performance of ID3 decision tree. Stage 4 patients can be stratified in significant groups characterized by the high confidence rules needed for making clinical decisions.
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- 2016
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50. Mechanisms of bradykinin-induced contraction in human fetal lung fibroblasts
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G. A. Rossi, Cesare Usai, Alessandra Eva, Cristina Vanni, Lm Fabbri, Federica Sabatini, Loredana Petecchia, Marzia Ognibene, Fabio Luigi Massimo Ricciardolo, and S Carnevali
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Pulmonary and Respiratory Medicine ,medicine.medical_specialty ,Time Factors ,Contraction (grammar) ,Myosin light-chain kinase ,Calmodulin ,Vasodilator Agents ,macromolecular substances ,Myosins ,Bradykinin ,Calcium in biology ,α-Smooth muscle actin * calcium * contraction * fibroblasts * myosin phosphorylation ,Contractility ,chemistry.chemical_compound ,Internal medicine ,Myosin ,medicine ,Humans ,Phosphorylation ,RNA, Small Interfering ,Fibroblast ,Lung ,biology ,Cell Differentiation ,Muscle, Smooth ,Fibroblasts ,Molecular biology ,Actins ,EGTA ,Endocrinology ,medicine.anatomical_structure ,Microscopy, Fluorescence ,chemistry ,biology.protein ,Collagen - Abstract
Bradykinin (BK) induces fibroblast contraction but the structural changes and intracellular mechanisms involved have not been completely explored. We stimulated HFL-1 fibroblasts with BK to assess: 1) fibroblast contractility; 2) the role of alpha-smooth muscle actin (SMA) in contraction by small interfering RNA (siRNA); 3) alpha-SMA protein expression; 4) alpha-SMA and F-actin structure; 5) intracellular calcium concentration; and 6) phosphorylated myosin light-chain (pMLC) and MLC kinase (MLCK) expression. BK triggered concentration- and time-dependent fibroblast gel contraction in conjunction with alpha-SMA over expression, but not in alpha-SMA-siRNA-treated cells. BK also increased alpha-SMA(+) and F-actin(+) cell number and stress fibre polymerisation (detectable at 5-60 min). These BK-induced changes were associated with an increase in intracellular calcium concentration, which peaked within 15 s, and activation of pMLC, which was detectable at 5-60 min. No MLCK content modification was observed. The different manifestations of the BK-induced fibroblast activation were downregulated at different levels (25-100%) by HOE140, a specific BK B2 receptor (B2R) antagonist and by the Ca(2+) chelator, EGTA. Thus, BK-induced fibroblast contraction, associated with differentiation into alpha-SMA(+) myofibroblasts, is mediated through the activation of the B2R and involves the Ca(2+)/calmodulin pMLC-dependent pathway.
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- 2010
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