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Ricercatore t.d. art. 24 c. 3 lett. B
Dipartimento di Scienze della Vita sede Centro di Medicina Rigenerativa

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2023 - Allele specific CRISPR/Cas9 editing of dominant Epidermolysis Bullosa Simplex in human epidermal stem cells [Articolo su rivista]
Cattaneo, C; Enzo, E; De Rosa, L; Sercia, L; Consiglio, F; Forcato, M; Bicciato, S; Paiardini, A; Basso, G; Tagliafico, E; Paganelli, A; Fiorentini, C; Magnoni, C; Latella, M C; De Luca, M

: Epidermolysis Bullosa Simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most of EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon1. Through the CRISPR/Cas9 system we performed a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

2023 - Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms [Abstract in Rivista]
Norfo, Ruggiero; Carretta, Chiara; Parenti, Sandra; Badii, Filippo; Bertesi, Matteo; Rontauroli, Sebastiano; Tavernari, Lara; Genovese, Elena; Sperduti, Samantha; Enzo, Elena; Mirabile, Margherita; Pedrazzi, Francesca; Pessina, Chiara; Colugnat, Ilaria; Mora, Barbara; Maccaferri, Monica; Tenedini, Elena; Martinelli, Silvia; Bianchi, Elisa; Casarini, Livio; Potenza, Leonardo; Luppi, Mario; Tagliafico, Enrico; Guglielmelli, Paola; Simoni, Manuela; Passamonti, Francesco; Vannucchi, Alessandro Maria; Manfredini, Rossella

2023 - Decoding the Human Epidermal Complexity at Single-Cell Resolution [Articolo su rivista]
Polito, MARIA PIA; Marini, Grazia; Palamenghi, Michele; Enzo, Elena

2022 - Clonal analysis of human clonogenic keratinocytes [Articolo su rivista]
Enzo, E.; Cattaneo, C.; Consiglio, F.; Polito, M. P.; Bondanza, S.; De Luca, M.

Regenerative medicine has its roots in harnessing stem cells for permanent restoration of damaged or diseased tissues. The first procedure for the transplantation of epidermal cultures in massive full-thickness burns was established in the 1980s. Since then, epithelial stem cell-based therapies have been further developed in cell and gene therapy protocols aimed at restoring visual acuity in severe ocular burns and treating patients affected by genetic skin diseases, as Epidermolysis Bullosa. The clinical success of these Advanced Therapy Medicinal Products (ATMPs) requires the presence of a defined number of epithelial stem cells in the grafts, detected as holoclone-forming cells. To date, the most trustworthy method to identify and measure holoclones in a culture is the clonal analysis of clonogenic keratinocytes. Here we describe in detail how to perform such a clonal analysis and identify each epidermal clonal type.

2022 - Genetic Disorders of the Extracellular Matrix: From Cell and Gene Therapy to Future Applications in Regenerative Medicine [Articolo su rivista]
Chakravarti, Shukti; Enzo, Elena; Rocha Monteiro de Barros, Maithê; Maffezzoni, Maria Benedetta Rizzarda; Pellegrini, Graziella

Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examinegenetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders.

2022 - Stairways to Advanced Therapies for Epidermolysis Bullosa [Articolo su rivista]
De Rosa, Laura; Enzo, Elena; Palamenghi, Michele; Sercia, Laura; De Luca, Michele

Epidermolysis bullosa (EB) is a devastating genetic skin disease typified by a plethora of different phenotypes and ranking from severe, early lethal, to mild localized forms. Although there is no cure for EB, recent progress in pharmacology and molecular and cellular biology is boosting the development of new advanced therapeutic strategies. Here we will focus on two main categories of such therapies: (1) those aimed at controlling inflammation and inducing reepithelialization of the wounds, and (2) those, perhaps more challenging and ambitious, that aim to permanently regenerate a fully functional epidermis, which requires targeting of epidermal stem cells. In both cases, the genetic variants underlying the different EB forms and factors, such as genetic background, modifier genes, comorbidities, and lifestyle, all of which impinge on EB genotype-phenotype correlation, need to be defined.

2021 - Hologene 5: A Phase II/III Clinical Trial of Combined Cell and Gene Therapy of Junctional Epidermolysis Bullosa [Articolo su rivista]
De Rosa, L.; Enzo, E.; Zardi, G.; Bodemer, C.; Magnoni, C.; Schneider, H.; De Luca, M.

Epidermolysis bullosa (EB) is a group of devastating genetic diseases characterized by skin and mucosal fragility and formation of blisters, which develop either spontaneously or in response to minor mechanical trauma. There is no definitive therapy for any form of EB. Intermediate junctional EB (JEB) caused by mutations in the gene LAMB3 has been the first genetic skin disease successfully tackled by ex vivo gene therapy. Here, we present a multicenter, open-label, uncontrolled phase II/III study that aims at confirming the efficacy of Hologene 5, a graft consisting of cultured transgenic keratinocytes and epidermal stem cells and meant to combine cell and gene therapy for the treatment of LAMB3-related JEB. Autologous clonogenic keratinocytes will be isolated from patients’ skin biopsies, genetically corrected with a gamma-retroviral vector (γRV) carrying the full-length human LAMB3 cDNA and plated onto a fibrin support (144cm2). The transgenic epidermis will be transplanted onto surgically prepared selected skin areas of at least six JEB patients (four pediatric and two adults). Evaluation of clinical efficacy will include, as primary endpoint, a combination of clinical parameters, such as percentage of re-epithelialization, cellular, molecular, and functional parameters, mechanical stress tests, and patient-reported outcome (PRO), up to 12months after transplantation. Safety and further efficacy endpoints will also be assessed during the clinical trial and for additional 15years in an interventional non-pharmacological follow-up study. If successful, this clinical trial would provide a therapeutic option for skin lesions of JEB patients with LAMB3 mutations and pave the way to a combined cell and gene therapy platform tackling other forms of EB and different genodermatoses. Clinical Trial Registration: EudraCT Number: 2018-000261-36.

2021 - Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells [Articolo su rivista]
Enzo, E.; Secone Seconetti, A.; Forcato, M.; Tenedini, E.; Polito, M. P.; Sala, I.; Carulli, S.; Contin, R.; Peano, C.; Tagliafico, E.; Bicciato, S.; Bondanza, S.; De Luca, M.

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

2019 - Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants [Articolo su rivista]
Salati, Simona; Genovese, Elena; Carretta, Chiara; Zini, Roberta; Bartalucci, Niccolò; Prudente, Zelia; Pennucci, Valentina; Ruberti, Samantha; Rossi, Chiara; Rontauroli, Sebastiano; Enzo, Elena; Calabresi, Laura; Balliu, Manjola; Mannarelli, Carmela; Bianchi, Elisa; Guglielmelli, Paola; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella

Somatic mutations of calreticulin (CALR) have been described in approximately 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.

2017 - Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells [Articolo su rivista]
Bauer, Johann; Koller, Josef; Murauer, Eva; DE ROSA, Laura; Enzo, Elena; Carulli, Sonia; Bondanza, Sergio; Recchia, Alessandra; Muss, Wolfgang; Diem, Anja; Mayr, Elisabeth; Schlager, Pamina; Gratz, Iris; Pellegrini, Graziella; DE LUCA, Michele

Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3,LAMB3,or LAMC2,which together encode laminin-332, a hetero-trimeric protein consisting ofa3,b3, andg2chain. In nonlethal generalized intermediate JEB, laminin-332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basement membrane upon minor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life. We report on a patient in whom gene-corrected epidermal sheets were transplanted onto a large nonhealing epidermal ulceration following a good manufacturing practice protocol

2017 - Regeneration of the entire human epidermis using transgenic stem cells [Articolo su rivista]
Hirsch, Tobias; Rothoeft, Tobias; Teig, Norbert; Bauer, Johann W.; Pellegrini, Graziella; De Rosa, Laura; Scaglione, Davide; Reichelt, Julia; Klausegger, Alfred; Kneisz, Daniela; Romano, Oriana; SECONE SECONETTI, Alessia; Contin, Roberta; Enzo, Elena; Jurman, Irena; Carulli, Sonia; Jacobsen, Frank; Luecke, Thomas; Lehnhardt, Marcus; Fischer, Meike; Kueckelhaus, Maximilian; Quaglino, Daniela; Morgante, Michele; Bicciato, Silvio; Bondanza, Sergio; De Luca, Michele

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, lifethreatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

2015 - Aerobic glycolysis tunes YAP/TAZ transcriptional activity [Articolo su rivista]
Enzo, Elena; Santinon, Giulia; Pocaterra, Arianna; Aragona, Mariaceleste; Bresolin, Silvia; Forcato, Mattia; Grifoni, Daniela; Pession, Annalisa; Zanconato, Francesca; Guzzo, Giulia; Bicciato, Silvio; Dupont, Sirio

Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro-tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth-promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro-tumorigenic activities such as YAP/TAZ.

2015 - The sweet side of YAP/TAZ [Articolo su rivista]
Santinon, G.; Enzo, E.; Dupont, S.

2014 - Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa. [Articolo su rivista]
DE ROSA, Laura; Carulli, S; Cocchiarella, Fabienne; Quaglino, Daniela; Enzo, Elena; Franchini, Eleonora; Giannetti, A; DE SANTIS, Giorgio; Recchia, Alessandra; Pellegrini, Graziella; DE LUCA, Michele

We report a long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, a devastating genetic skin disease. The critical goals of the trial were to evaluate the safety and long-term persistence of genetically modified epidermis. A normal epidermal-dermal junction was restored and the regenerated transgenic epidermis was found to be fully functional and virtually indistinguishable from a normal control. The epidermis was sustained by a discrete number of long-lasting, self-renewing transgenic epidermal stem cells that maintained the memory of the donor site, whereas the vast majority of transduced transit-amplifying progenitors were lost within the first few months after grafting. These data pave the way for the safe use of epidermal stem cells in combined cell and gene therapy for genetic skin diseases.

2014 - YAP/TAZ Incorporation in the β-Catenin Destruction Complex Orchestrates the Wnt Response. [Articolo su rivista]
Azzolin, L; Panciera, T; Soligo, S; Enzo, Elena; Bicciato, Silvio; Dupont, S; Bresolin, S; Frasson, C; Basso, G; Guzzardo, V; Fassina, A; Cordenonsi, M; Piccolo, S.

The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the β-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for β-TrCP recruitment to the complex and β-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/β-catenin signaling. In line, the β-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression

2013 - BMP signaling controls muscle mass [Articolo su rivista]
Sartori, R.; Schirwis, E.; Blaauw, B.; Bortolanza, S.; Zhao, J.; Enzo, E.; Stantzou, A.; Mouisel, E.; Toniolo, L.; Ferry, A.; Stricker, S.; Goldberg, A. L.; Dupont, S.; Piccolo, S.; Amthor, H.; Sandri, M.

Cell size is determined by the balance between protein synthesis and degradation. This equilibrium is affected by hormones, nutrients, energy levels, mechanical stress and cytokines. Mutations that inactivate myostatin lead to excessive muscle growth in animals and humans, but the signals and pathways responsible for this hypertrophy remain largely unknown. Here we show that bone morphogenetic protein (BMP) signaling, acting through Smad1, Smad5 and Smad8 (Smad1/5/8), is the fundamental hypertrophic signal in mice. Inhibition of BMP signaling causes muscle atrophy, abolishes the hypertrophic phenotype of myostatin-deficient mice and strongly exacerbates the effects of denervation and fasting. BMP-Smad1/5/8 signaling negatively regulates a gene (Fbxo30) that encodes a ubiquitin ligase required for muscle loss, which we named muscle ubiquitin ligase of the SCF complex in atrophy-1 (MUSA1). Collectively, these data identify a critical role for the BMP pathway in adult muscle maintenance, growth and atrophy.

2013 - P63, Sharp1, and HIFs: Master regulators of metastasis in triple-negative breast cance [Articolo su rivista]
Piccolo, S.; Enzo, E.; Montagner, M.

Metastasis is the most significant cause of cancer-associated morbidity and mortality but remains poorly understood. Recent work revealed that metastasis of aggressive triple-negative breast cancers is suppressed by Sharp1, a factor that promotes degradation of hypoxia-inducible factors (HIF) and blunts HIF-induced malignant cell behavior. (C) 2013 AACR.

2012 - Fat facets deubiquitylation of Medea/Smad4 modulates interpretation of a Dpp morphogen gradient [Articolo su rivista]
Stinchfield, M. J.; Takaesu, N. T.; Quijano, J. C.; Castillo, A. M.; Tiusanen, N.; Shimmi, O.; Enzo, E.; Dupont, S.; Piccolo, S.; Newfeld, S. J.

The ability of secreted Transforming Growth Factor beta (TGF beta) proteins to act as morphogens dictates that their influence be strictly regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control of cellular responsiveness to TGF beta signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper implementation of a morphogen gradient.

2012 - Self-regulation of the head-inducing properties of the Spemann organizer [Articolo su rivista]
Inui, M.; Montagner, M.; Ben-Zvi, D.; Martello, G.; Soligo, S.; Manfrin, A.; Aragona, M.; Enzo, E.; Zacchigna, L.; Zanconato, F.; Azzolin, L.; Dupont, S.; Cordenonsi, M.; Piccolo, S.

The Spemann organizer stands out from other signaling centers of the embryo because of its broad patterning effects. It defines development along the anteroposterior and dorsoventral axes of the vertebrate body, mainly by secreting antagonists of growth factors. Qualitative models proposed more than a decade ago explain the organizer's region-specific inductions (i.e., head and trunk) as the result of different combinations of antagonists. For example, head induction is mediated by extracellular inhibition of Wnt, BMP, and Nodal ligands. However, little is known about how the levels of these antagonists become harmonized with those of their targets and with the factors initially responsible for germ layers and organizer formation, including Nodal itself. Here we show that key ingredients of the head-organizer development, namely Nodal ligands, Nodal antagonists, and ADMP ligands reciprocally adjust each other's strength and range of activity by a self-regulating network of interlocked feedback and feedforward loops. A key element in this cross-talk is the limited availability of ACVR2a, for which Nodal and ADMP must compete. By trapping Nodal extracellularly, the Nodal antagonists Cerberus and Lefty are permissive for ADMP activity. The system self-regulates because ADMP/ACVR2a/Smad1 signaling in turn represses the expression of the Nodal antagonists, reestablishing the equilibrium. In sum, this work reveals an unprecedented set of interactions operating within the organizer that is critical for embryonic patterning.

2012 - SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors [Articolo su rivista]
Montagner, M; Enzo, Elena; Forcato, Mattia; Zanconato, F; Parenti, A; Rampazzo, E; Basso, G; Leo, G; Rosato, A; Bicciato, Silvio; Cordenonsi, M; Piccolo, S.

The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel-Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.

2012 - Signaling crosstalk between TGFβ and Dishevelled/Par1b [Articolo su rivista]
Mamidi, A.; Inui, M.; Manfrin, A.; Soligo, S.; Enzo, E.; Aragona, M.; Cordenonsi, M.; Wessely, O.; Dupont, S.; Piccolo, S.

Crosstalk of signaling pathways is critical during metazoan development and adult tissue homeostasis. Even though the transforming growth factor-beta (TGF beta) transduction cascade is rather simple, in vivo responsiveness to TGF beta ligands is tightly regulated at several steps. As such, TGF beta represents a paradigm for how the activity of one signaling system is modulated by others. Here, we report an unsuspected regulatory step involving Dishevelled (Dvl) and Par1b (also known as MARK2). Dvl and Par1b cooperate to enable TGF beta/bone morphogenetic protein (BMP) signaling in Xenopus mesoderm development and TGF beta responsiveness in mammalian cells. Mechanistically, the assembly of the Par1b/Dvl3/Smad4 complex is fostered by Wnt5a. The association of Smad4 to Dvl/Par1 prevents its inhibitory ubiquitination by ectodermin (also known as transcriptional intermediary factor 1 gamma or tripartite motif protein 33). We propose that this crosstalk is relevant to coordinate TGF beta responses with Wnt-noncanonical and polarity pathways. Cell Death and Differentiation (2012) 19, 1689-1697; doi:10.1038/cdd.2012.50; published online 11 May 2012

2011 - Role of YAP/TAZ in mechanotransduction [Articolo su rivista]
Dupont, S; Morsut, L; Aragona, M; Enzo, Elena; Giulitti, S; Cordenonsi, M; Zanconato, F; Le Digabel, J; Forcato, Mattia; Bicciato, Silvio; Elvassore, N; Piccolo, S.

Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment

2011 - USP15 is a deubiquitylating enzyme for receptor-activated SMADs [Articolo su rivista]
Inui, M.; Manfrin, A.; Mamidi, A.; Martello, G.; Morsut, L.; Soligo, S.; Enzo, E.; Moro, S.; Polo, S.; Dupont, S.; Cordenonsi, M.; Piccolo, S.

The TGF beta pathway is critical for embryonic development and adult tissue homeostasis. On ligand stimulation, TGF beta and BMP receptors phosphorylate receptor-activated SMADs (R-SMADs), which then associate with SMAD4 to form a transcriptional complex that regulates gene expression through specific DNA recognition(1,2). Several ubiquitin ligases serve as inhibitors of R-SMADs(3,4), yet no deubiquitylating enzyme (DUB) for these molecules has so far been identified. This has left unexplored the possibility that ubiquitylation of R-SMADs is reversible and engaged in regulating SMAD function, in addition to degradation(5). Here we identify USP15 as a DUB for R-SMADs. USP15 is required for TGF beta and BMP responses in mammalian cells and Xenopus embryos. At the biochemical level, USP15 primarily opposes R-SMAD monoubiquitylation, which targets the DNA-binding domains of R-SMADs and prevents promoter recognition. As such, USP15 is critical for the occupancy of endogenous target promoters by the SMAD complex. These data identify an additional layer of control by which the ubiquitin system regulates TGF beta biology.

2010 - A MicroRNA targeting dicer for metastasis control [Articolo su rivista]
Martello, G; Rosato, A; Ferrari, F; Manfrin, A; Cordenonsi, M; Dupont, S; Enzo, Elena; Guzzardo, V; Rondina, M; Spruce, T; Parenti, Ar; Daidone, Mg; Bicciato, Silvio; Piccolo, S.

Although specific microRNAs (miRNAs) can be upregulated in cancer, global miRNA downregulation is a common trait of human malignancies. The mechanisms of this phenomenon and the advantages it affords remain poorly understood. Here we identify a microRNA family, miR-103/107, that attenuates miRNA biosynthesis by targeting Dicer, a key component of the miRNA processing machinery. In human breast cancer, high levels of miR-103/107 are associated with metastasis and poor outcome. Functionally, miR-103/107 confer migratory capacities in vitro and empower metastatic dissemination of otherwise nonaggressive cells in vivo. Inhibition of miR-103/107 opposes migration and metastasis of malignant cells. At the cellular level, a key event fostered by miR-103/107 is induction of epithelial-to-mesenchymal transition (EMT), attained by downregulating miR-200 levels. These findings suggest a new pathway by which Dicer inhibition drifts epithelial cancer toward a less-differentiated, mesenchymal fate to foster metastasis

2010 - Negative control of Smad activity by ectodermin/Tif1γ patterns the mammalian embryo [Articolo su rivista]
Morsut, L.; Yan, K. -P.; Enzo, E.; Aragona, M.; Soligo, S. M.; Wendling, O.; Mark, M.; Khetchoumian, K.; Bressan, G.; Chambon, P.; Dupont, S.; Losson, R.; Piccolo, S.

The definition of embryonic potency and induction of specific cell fates are intimately linked to the tight control over TGF beta signaling. Although extracellular regulation of ligand availability has received considerable attention in recent years, surprisingly little is known about the intracellular factors that negatively control Smad activity in mammalian tissues. By means of genetic ablation, we show that the Smad4 inhibitor ectodermin (Ecto, also known as Trim33 or Tif1 gamma) is required to limit Nodal responsiveness in vivo. New phenotypes, which are linked to excessive Nodal activity, emerge from such a modified landscape of Smad responsiveness in both embryonic and extra-embryonic territories. In extra-embryonic endoderm, Ecto is required to confine expression of Nodal antagonists to the anterior visceral endoderm. In trophoblast cells, Ecto precisely doses Nodal activity, balancing stem cell self-renewal and differentiation. Epiblast-specific Ecto deficiency shifts mesoderm fates towards node/organizer fates, revealing the requirement of Smad inhibition for the precise allocation of cells along the primitive streak. This study unveils that intracellular negative control of Smad function by ectodermin/Tif1 gamma is a crucial element in the cellular response to TGF beta signals in mammalian tissues.