title: Hyper-activation of Notch3 amplifies the proliferative potential of rhabdomyosarcoma cells.
abstract: Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: De Salvo
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Lavinia
authors.initials: L
authors.lastname: Raimondi
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Vella
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Adesso
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Ciarapica
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Verginelli
authors.affiliation: Stanley Scott Cancer Center, Louisiana State University Health Sciences Center and Louisiana Cancer Research Consortium, New Orleans, Louisiana, United States of America.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pannuti
authors.affiliation: Department of Pathology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Arianna
authors.initials: A
authors.lastname: Citti
authors.affiliation: Department of Pathology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Renata
authors.initials: R
authors.lastname: Boldrini
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Giuseppe M
authors.initials: GM
authors.lastname: Milano
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Cacchione
authors.affiliation: Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ferrari
authors.affiliation: Anatomic Pathology Unit 2, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Collini
authors.affiliation: Department of Pediatrics, Oncohematology Unit, University of Padova, Padova, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Rosolen
authors.affiliation: Department of Pediatrics, Oncohematology Unit, University of Padova, Padova, Italy.
authors.forename: Gianni
authors.initials: G
authors.lastname: Bisogno
authors.affiliation: Department of Pathology, University of Padova, Padova, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Alaggio
authors.affiliation: Department of Surgery, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Inserra
authors.affiliation: Department of Scientific Directorate, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Mattia
authors.initials: M
authors.lastname: Locatelli
authors.affiliation: Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors.forename: Stefano
authors.initials: S
authors.lastname: Stifani
authors.affiliation: Department of Molecular Medicine, Sapienza University, Roma, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Screpanti
authors.affiliation: Stanley Scott Cancer Center, Louisiana State University Health Sciences Center and Louisiana Cancer Research Consortium, New Orleans, Louisiana, United States of America.
authors.forename: Lucio
authors.initials: L
authors.lastname: Miele
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy; Dipartimento di Scienze Pediatriche, Università di Pavia, Pavia, Italy.
authors.forename: Franco
authors.initials: F
authors.lastname: Locatelli
authors.affiliation: Department of Oncohematology, Ospedale Pediatrico Bambino Gesù, IRCCS, Roma, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Rota
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000096:biosynthesis / Q000235:genetics; D051761:Paired Box Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D018232:Rhabdomyosarcoma, Alveolar / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018233:Rhabdomyosarcoma, Embryonal / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction
chemical_list: D019394:Ki-67 Antigen; C497761:NOTCH1 protein, human; C499374:NOTCH3 protein, human; D015514:Oncogene Proteins, Fusion; C471563:PAX3-FOXO1A fusion protein, human; D051761:Paired Box Transcription Factors; D051881:Receptor, Notch1; D000071656:Receptor, Notch3; D051880:Receptors, Notch