Elena Casanova

Scopus Publications

Protocol for in vitro establishment of heterogeneous stem-like cultures derived from whole human glioblastoma tumors
Francesca De Bacco, Francesca Orzan, Elena Casanova, Marta Prelli, Carla Boccaccio
STAR Protocols, 2023
Cultures enriched in glioblastoma stem-like cells (GSCs) are prominent in vitro models to investigate molecular determinants and therapeutic targets of glioblastoma; however, conventional GSC derivation protocols fail to preserve GSC heterogeneity. Here, we present a protocol for the propagation of heterogeneous GSC cultures starting from cell resuspensions containing the entire tumor mass. We describe steps for isolation of GSCs and their maintenance and expansion in culture. We then detail procedures for preliminary analysis to be performed on freshly isolated material. For complete details on the use and execution of this protocol, please refer to De Bacco et al.1
Coexisting cancer stem cells with heterogeneous gene amplifications, transcriptional profiles, and malignancy are isolated from single glioblastomas
Francesca De Bacco, Francesca Orzan, Giovanni Crisafulli, Marta Prelli, Claudio Isella, Elena Casanova, Raffaella Albano, Gigliola Reato, Jessica Erriquez, Antonio D’Ambrosio, Mara Panero, Carmine Dall’Aglio, Laura Casorzo, Manuela Cominelli, Francesca Pagani, Antonio Melcarne, Pietro Zeppa, Roberto Altieri, Isabella Morra, Paola Cassoni, Diego Garbossa, Anna Cassisa, Alice Bartolini, Serena Pellegatta, Paolo M. Comoglio, Gaetano Finocchiaro, Pietro L. Poliani, Carla Boccaccio
Cell Reports, 2023
Glioblastoma (GBM) is known as an intractable, highly heterogeneous tumor encompassing multiple subclones, each supported by a distinct glioblastoma stem cell (GSC). The contribution of GSC genetic and transcriptional heterogeneity to tumor subclonal properties is debated. In this study, we describe the systematic derivation, propagation, and characterization of multiple distinct GSCs from single, treatment-naive GBMs (GSC families). The tumorigenic potential of each GSC better correlates with its transcriptional profile than its genetic make-up, with classical GSCs being inherently more aggressive and mesenchymal more dependent on exogenous growth factors across multiple GBMs. These GSCs can segregate and recapitulate different histopathological aspects of the same GBM, as shown in a paradigmatic tumor with two histopathologically distinct components, including a conventional GBM and a more aggressive primitive neuronal component. This study provides a resource for investigating how GSCs with distinct genetic and/or phenotypic features contribute to individual GBM heterogeneity and malignant escalation.
TFEB inhibition induces melanoma shut-down by blocking the cell cycle and rewiring metabolism
C. Ariano, F. Costanza, M. Akman, C. Riganti, D. Corà, E. Casanova, E. Astanina, V. Comunanza, F. Bussolino, G. Doronzo
Cell Death and Disease, 2023
Melanomas are characterised by accelerated cell proliferation and metabolic reprogramming resulting from the contemporary dysregulation of the MAPK pathway, glycolysis and the tricarboxylic acid (TCA) cycle. Here, we suggest that the oncogenic transcription factor EB (TFEB), a key regulator of lysosomal biogenesis and function, controls melanoma tumour growth through a transcriptional programme targeting ERK1/2 activity and glucose, glutamine and cholesterol metabolism. Mechanistically, TFEB binds and negatively regulates the promoter of DUSP-1, which dephosphorylates ERK1/2. In melanoma cells, TFEB silencing correlates with ERK1/2 dephosphorylation at the activation-related p-Thr185 and p-Tyr187 residues. The decreased ERK1/2 activity synergises with TFEB control of CDK4 expression, resulting in cell proliferation blockade. Simultaneously, TFEB rewires metabolism, influencing glycolysis, glucose and glutamine uptake, and cholesterol synthesis. In TFEB-silenced melanoma cells, cholesterol synthesis is impaired, and the uptake of glucose and glutamine is inhibited, leading to a reduction in glycolysis, glutaminolysis and oxidative phosphorylation. Moreover, the reduction in TFEB level induces reverses TCA cycle, leading to fatty acid production. A syngeneic BRAFV600E melanoma model recapitulated the in vitro study results, showing that TFEB silencing sustains the reduction in tumour growth, increase in DUSP-1 level and inhibition of ERK1/2 action, suggesting a pivotal role for TFEB in maintaining proliferative melanoma cell behaviour and the operational metabolic pathways necessary for meeting the high energy demands of melanoma cells.
The PSI Domain of the MET Oncogene Encodes a Functional Disulfide Isomerase Essential for the Maturation of the Receptor Precursor
Dogus Murat Altintas, Simona Gallo, Cristina Basilico, Marina Cerqua, Alessio Bocedi, Annapia Vitacolonna, Orsola Botti, Elena Casanova, Ilaria Rancati, Chiara Milanese, Sara Notari, Giorgia Gambardella, Giorgio Ricci, Pier Giorgio Mastroberardino, Carla Boccaccio, Tiziana Crepaldi, Paolo Maria Comoglio
International Journal of Molecular Sciences, 2022
The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of α and β chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand—hepatocyte growth factor (HGF)—such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein.
Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor
Claudia Desole, Simona Gallo, Annapia Vitacolonna, Elisa Vigna, Cristina Basilico, Francesca Montarolo, Francesca Zuppini, Elena Casanova, Riccardo Miggiano, Davide Maria Ferraris, Antonio Bertolotto, Paolo Maria Comoglio, Tiziana Crepaldi
Frontiers in Immunology, 2021
The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.
ERBB3 overexpression due to miR-205 inactivation confers sensitivity to FGF, metabolic activation, and liability to ERBB3 targeting in glioblastoma
Francesca De Bacco, Francesca Orzan, Jessica Erriquez, Elena Casanova, Ludovic Barault, Raffaella Albano, Antonio D’Ambrosio, Viola Bigatto, Gigliola Reato, Monica Patanè, Bianca Pollo, Geoffrey Kuesters, Carmine Dell’Aglio, Laura Casorzo, Serena Pellegatta, Gaetano Finocchiaro, Paolo M. Comoglio, Carla Boccaccio
Cell Reports, 2021
In glioblastoma (GBM), the most frequent and lethal brain tumor, therapies suppressing recurrently altered signaling pathways failed to extend survival. However, in patient subsets, specific genetic lesions can confer sensitivity to targeted agents. By exploiting an integrated model based on patient-derived stem-like cells, faithfully recapitulating the original GBMs in vitro and in vivo, here, we identify a human GBM subset (∼9% of all GBMs) characterized by ERBB3 overexpression and nuclear accumulation. ERBB3 overexpression is driven by inheritable promoter methylation or post-transcriptional silencing of the oncosuppressor miR-205 and sustains the malignant phenotype. Overexpressed ERBB3 behaves as a specific signaling platform for fibroblast growth factor receptor (FGFR), driving PI3K/AKT/mTOR pathway hyperactivation, and overall metabolic upregulation. As a result, ERBB3 inhibition by specific antibodies is lethal for GBM stem-like cells and xenotransplants. These findings highlight a subset of patients eligible for ERBB3-targeted therapy.
The long-lasting protective effect of HGF in cardiomyoblasts exposed to doxorubicin requires a positive feed-forward loop mediated by ERK1,2-TIMP1-STAT3
Simona Gallo, Martina Spilinga, Elena Casanova, Alessandro Bonzano, Carla Boccaccio, Paolo Maria Comoglio, Tiziana Crepaldi
International Journal of Molecular Sciences, 2020
Previous studies showed that the hepatocyte growth factor (HGF)–Met receptor axis plays long-lasting cardioprotection against doxorubicin anti-cancer therapy. Here, we explored the mechanism(s) underlying the HGF protective effect. DNA damage was monitored by histone H2AX phosphorylation and apoptosis by proteolytic cleavage of caspase 3. In doxorubicin-treated H9c2 cardiomyoblasts, the long-lasting cardioprotection is mediated by activation of the Ras/Raf/Mek/Erk (extracellular signal-regulated kinase 1,2) signaling pathway and requires Stat3 (signal transducer and activator of transcription 3) activation. The HGF protection was abrogated by the Erk1,2 inhibitor, PD98059. This translated into reduced Y705 phosphorylation and impaired nuclear translocation of Stat3, showing crosstalk between Erk1,2 and Stat3 signaling. An array of 29 cytokines, known to activate Stat3, was interrogated to identify the molecule(s) linking the two pathways. The analysis showed a selective increase in expression of the tissue inhibitor of metalloproteinases-1 (Timp1). Consistently, inhibition in cardiomyoblasts of Timp1 translation by siRNAs blunted both Stat3 activation and the cardioprotective effect of HGF. Thus, Timp1 is responsible for the generation of a feed-forward loop of Stat3 activation and helps cardiomyocytes to survive during the genotoxic stress induced by anthracyclines.
Activation of the MET receptor attenuates doxorubicin-induced cardiotoxicity in vivo and in vitro
Simona Gallo, Martina Spilinga, Raffaella Albano, Giuseppe Ferrauto, Enza Di Gregorio, Elena Casanova, Davide Balmativola, Alessandro Bonzano, Carla Boccaccio, Anna Sapino, Paolo Maria Comoglio, Tiziana Crepaldi
British Journal of Pharmacology, 2020
Doxorubicin anti‐cancer therapy is associated with cardiotoxicity, resulting from DNA damage response (DDR). Hepatocyte growth factor (HGF) protects cardiomyocytes from injury, but its effective use is compromised by low biodistribution. In this study, we have investigated whether the activation of the HGF receptor—encoded by the Met gene—by an agonist monoclonal antibody (mAb) could protect against doxorubicin‐induced cardiotoxicity.
MET inhibition overcomes radiation resistance of glioblastoma stem-like cells
Francesca De Bacco, Antonio D'Ambrosio, Elena Casanova, Francesca Orzan, Roberta Neggia, Raffaella Albano, Federica Verginelli, Manuela Cominelli, Pietro L Poliani, Paolo Luraghi, Gigliola Reato, Serena Pellegatta, Gaetano Finocchiaro, Timothy Perera, Elisabetta Garibaldi, Pietro Gabriele, Paolo M Comoglio, Carla Boccaccio
EMBO Molecular Medicine, 2016
Glioblastoma (GBM) contains stem‐like cells (GSCs) known to be resistant to ionizing radiation and thus responsible for therapeutic failure and rapidly lethal tumor recurrence. It is known that GSC radioresistance relies on efficient activation of the DNA damage response, but the mechanisms linking this response with the stem status are still unclear. Here, we show that the MET receptor kinase, a functional marker of GSCs, is specifically expressed in a subset of radioresistant GSCs and overexpressed in human GBM recurring after radiotherapy. We elucidate that MET promotes GSC radioresistance through a novel mechanism, relying on AKT activity and leading to (i) sustained activation of Aurora kinase A, ATM kinase, and the downstream effectors of DNA repair, and (ii) phosphorylation and cytoplasmic retention of p21, which is associated with anti‐apoptotic functions. We show that MET pharmacological inhibition causes DNA damage accumulation in irradiated GSCs and their depletion in vitro and in GBMs generated by GSC xenotransplantation. Preclinical evidence is thus provided that MET inhibitors can radiosensitize tumors and convert GSC‐positive selection, induced by radiotherapy, into GSC eradication.
TNF-α promotes invasive growth through the MET signaling pathway
Viola Bigatto, Francesca De Bacco, Elena Casanova, Gigliola Reato, Letizia Lanzetti, Claudio Isella, Ivana Sarotto, Paolo M. Comoglio, Carla Boccaccio
Molecular Oncology, 2015
The inflammatory cytokine Tumor Necrosis Factor Alpha (TNF-α) is known to trigger invasive growth, a physiological property for tissue healing, turning into a hallmark of progression in cancer. However, the invasive response to TNF-α relies on poorly understood molecular mechanisms. We thus investigated whether it involves the MET oncogene, which regulates the invasive growth program by encoding the tyrosine kinase receptor for Hepatocyte Growth Factor (HGF). Here we show that the TNF-α pro-invasive activity requires MET function, as it is fully inhibited by MET-specific inhibitors (small-molecules, antibodies, and siRNAs). Mechanistically, we show that TNF-α induces MET transcription via NF-κB, and exploits MET to sustain MEK/ERK activation and Snail accumulation, leading to E-cadherin downregulation. We then show that TNF-α not only induces MET expression in cancer cells, but also HGF secretion by fibroblasts. Consistently, we found that, in human colorectal cancer tissues, high levels of TNF-α correlates with increased expression of both MET and HGF. These findings suggest that TNF-α fosters a HGF/MET pro-invasive paracrine loop in tumors. Targeting this ligand/receptor pair would contribute to prevent cancer progression associated with inflammation.
Agonist antibodies activating the Met receptor protect cardiomyoblasts from cobalt chloride-induced apoptosis and autophagy
S Gallo, S Gatti, V Sala, R Albano, P Costelli, E Casanova, P M Comoglio, T Crepaldi
Cell Death and Disease, 2014
The MET oncogene is a functional marker of a glioblastoma stem cell subtype
Francesca De Bacco, Elena Casanova, Enzo Medico, Serena Pellegatta, Francesca Orzan, Raffaella Albano, Paolo Luraghi, Gigliola Reato, Antonio D'Ambrosio, Paola Porrati, Monica Patanè, Emanuela Maderna, Bianca Pollo, Paolo M. Comoglio, Gaetano Finocchiaro, Carla Boccaccio
Cancer Research, 2012

Elena Casanova

EDUCATION

RESEARCH, TEACHING, or OTHER INTERESTS

Scopus Publications