Abstract 4880: Regulation of extracellular microvesicles secretion by Rab27b in glioblastoma multiforme
Authors: Rodrigues Bruna R. , Giudice Fernanda S. , Dias Marcos S. , Baptistella Antuani R. , Malagoli Rafael R. , Sanematsu Paulo I. , Suzuki Sergio , Reis Mariana , Martins Vilma R. 
Published: May 2015

Abstract
Background: Glioblastoma multiforme (GBM) is the most malignant form of astrocytomas and they release a heterogeneous population of microvesicles (MVEs) to the extracellular space. These MVEs are involved in intercellular communication by carrying oncogenes, and miRNAs that contribute with the tumor aggressiveness. However, the mechanisms of MVEs biogenesis and secretion in GBM are poorly understood.
Material and Methods: Using five glioblastoma cell lines (LN-18, LN-229, U87MG, A-172 and U118MG) the vesicular biogenesis was characterize analyzing the expression of markers involved in steps of this process by western blotting. MVEs were also quantified using Nanoparticle Tracking Analysis (NTA - NanoSight®). Immunofluorescence was performed in these cell lines to characterize the cellular distribution of Rab27b, which is responsible for driving the multivesicular bodies to the plasma membrane. Immunohistochemistry (IHC) measured Rab27b expression in a tissue microarray with 60 GBMs samples and 13 non-neoplastic human brain tissues used as controls. IHC intensity was scored from 1 to 3 (1 weak; 2, moderate; 3, strong) and positive cells, from 0 to 3 (0, 0%; 1, 1-10%+; 2, 11-50%+; 3, ≥ 51%+). The final IHC score was represented by intensity + positivity. Student's t-test was performed to compare Rab27b expression in GBM and non neoplastic tissues. Kaplan-Meier and log-rank tests were used to correlate overall survival with Rab27b expression.
Results: The cell lines showed a heterogeneous expression of markers involved in vesicle biogenesis. LN-18 cells presented higher levels of Cavelin-1, Flotillin -1, Alix and VPS4, p&lt;0.01, but lower levels of Rab27b, p&lt;0.0001. On the other hand, the U87MG cells standed out by higher levels of Rab27b, compared to the other cell lines studied, p&lt;0.0001. Interesting, our results pointed that MVEs secretion is directed correlated to the expression pattern of Rab27b in GBM cells. Indeed, LN-18 cells showed lower levels of MVEs secretion (p&lt;0.001) while U87MG cells presented higher MVEs secretion, p&lt;0.001. In addition, Rab27b and CD63 (MVEs marker) were retained in the perinuclear region of LN -18 cells while in U87MG cells both proteins were distributed throughout the cytoplasm. Finally, Rab27b is overexpressed in GBM samples when compared to non-neoplastic tissue and patients presenting high expression of Rab27b showed lower survival outcomes.
Conclusions: Our findings suggest that Rab27b may be crucial to the secretion of MVEs process in GBM. Furthermore, the overexpression of Rab27b in glioblastoma patients coincides with a poor prognostic phenotype.
Supported by São Paulo State Foundation (FAPESP).
Citation Format: Bruna R. Rodrigues, Fernanda S. Giudice, Marcos S. Dias, Antuani R. Baptistella, Rafael R. Malagoli, Paulo I. Sanematsu, Sergio Suzuki, Mariana Reis, Vilma R. Martins. Regulation of extracellular microvesicles secretion by Rab27b in glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4880. doi:10.1158/1538-7445.AM2014-4880