title: The availability of the embryonic TGF-β protein Nodal is dynamically regulated during glioblastoma multiforme tumorigenesis.
abstract: BACKGROUND
  Glioblastoma (GBM) is the most common primary brain tumor presenting self-renewing cancer stem cells. The role of these cells on the development of the tumors has been proposed to recapitulate programs from embryogenesis. Recently, the embryonic transforming growth factor-β (TGF-β) protein Nodal has been shown to be reactivated upon tumor development; however, its availability in GBM cells has not been addressed so far. In this study, we investigated by an original approach the mechanisms that dynamically control both intra and extracellular Nodal availability during GBM tumorigenesis.
  
  
  METHODS
  We characterized the dynamics of Nodal availability in both stem and more differentiated GBM cells through morphological analysis, immunofluorescence of Nodal protein and of early (EEA1 and Rab5) and late (Rab7 and Rab11) endocytic markers and Western Blot. Tukey's test was used to analyze the prevalent correlation of Nodal with different endocytic markers inside specific differentiation states, and Sidak's multiple comparisons test was used to compare the prevalence of Nodal/endocytic markers co-localization between two differentiation states of GBM cells. Paired t test was used to analyze the abundance of Nodal protein, in extra and intracellular media.
  
  
  RESULTS
  The cytoplasmic distribution of Nodal was dynamically regulated and strongly correlated with the differentiation status of GBM cells. While Nodal-positive vesicle-like particles were symmetrically distributed in GBM stem cells (GBMsc), they presented asymmetric perinuclear localization in more differentiated GBM cells (mdGBM). Strikingly, when subjected to dedifferentiation, the distribution of Nodal in mdGBM shifted to a symmetric pattern. Moreover, the availability of both intracellular and secreted Nodal were downregulated upon GBMsc differentiation, with cells becoming elongated, negative for Nodal and positive for Nestin. Interestingly, the co-localization of Nodal with endosomal vesicles also depended on the differentiation status of the cells, with Nodal seen more packed in EEA1/Rab5 + vesicles in GBMsc and more in Rab7/11 + vesicles in mdGBM.
  
  
  CONCLUSIONS
  Our results show for the first time that Nodal availability relates to GBM cell differentiation status and that it is dynamically regulated by an endocytic pathway during GBM tumorigenesis, shedding new light on molecular pathways that might emerge as putative targets for Nodal signaling in GBM therapy.
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F2-01, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: Maria Cecília
authors.initials: MC
authors.lastname: Oliveira-Nunes
authors.affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 265 Campus Drive, Stanford, California 94305 USA ; Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F1-20, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: Suzana
authors.initials: S
authors.lastname: Assad Kahn
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F2-01, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: Ana Luiza
authors.initials: AL
authors.lastname: de Oliveira Barbeitas
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F1-20, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil ; Instituto Estadual do Cérebro Paulo Niemeyer (IECPN), Rua do Rezende, 156, Rio de Janeiro, Rio de Janeiro 20231-092 Brazil.
authors.forename: Tania Cristina Leite de Sampaio
authors.initials: TC
authors.lastname: E Spohr
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F1-20, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil ; Instituto Estadual do Cérebro Paulo Niemeyer (IECPN), Rua do Rezende, 156, Rio de Janeiro, Rio de Janeiro 20231-092 Brazil.
authors.forename: Luiz Gustavo Feijó
authors.initials: LG
authors.lastname: Dubois
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, B1-29, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: Grasiella Maria
authors.initials: GM
authors.lastname: Ventura Matioszek
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F2-30, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: William
authors.initials: W
authors.lastname: Querido
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F1-20, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: Loraine
authors.initials: L
authors.lastname: Campanati
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F2-01, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: José Marques
authors.initials: JM
authors.lastname: de Brito Neto
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F1-20, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: Flavia Regina Souza
authors.initials: FR
authors.lastname: Lima
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F1-20, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil ; Instituto Estadual do Cérebro Paulo Niemeyer (IECPN), Rua do Rezende, 156, Rio de Janeiro, Rio de Janeiro 20231-092 Brazil.
authors.forename: Vivaldo
authors.initials: V
authors.lastname: Moura-Neto
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Av. Carlos Chagas Filho 373, F2-01, Rio de Janeiro, Rio de Janeiro 21941-902 Brazil.
authors.forename: Katia
authors.identifier: 0000-0002-2006-9342
authors.initials: K
authors.lastname: Carneiro
keywords: Cancer stem-cell; Endocytosis; Glioblastoma; Nodal; Tumorigenesis