1472 Proteomics 2014, 14, 1472–1479 DOI 10.1002/pmic.201300485 RESEARCH ARTICLE The overexpression of a single oncogene (ERBB2/HER2) alters the proteomic landscape of extracellular vesicles Maria Amorim 1,2 , Gustavo Fernandes 1 , Paulo Oliveira 3 , Daniel Martins-de-Souza 4,5∗ , Emmanuel Dias-Neto 1,4∗ and Diana Nunes 1 1 Laboratory of Medical Genomics, AC Camargo Cancer Center, S˜ ao Paulo, SP, Brazil 2 P´ os-graduac ¸˜ ao em Oncologia, Fundac ¸˜ ao Ant ˆ onio Prudente, S ˜ ao Paulo, SP, Brazil 3 Laborat ´ orio Nacional de Bioci ˆ encias (LNBio), Campinas, SP, Brazil 4 Laborat ´ orio de Neuroci ˆ encias Alzira Denise Hertzog Silva (LIM27), Instituto de Psiquiatria, FMUSP, S˜ ao Paulo, SP, Brazil 5 Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University Munich, Munich, Germany Received: November 2, 2013 Revised: March 7, 2014 Accepted: March 27, 2014 ERBB2/HER2 amplification activates signaling cascades that lead to a tumor cell phenotype. However, despite its remarkable importance in oncology, the consequences of HER2 ampli- fication over the extracellular vesicles (EVs) content have not yet been investigated. Here, we isolated EVs secreted by HB4a, a mammary luminal epithelial cell line and C5.2, its HER2- overexpressing clone. We isolated two EV sets (20 and 100 K) by ultracentrifugation and used electron microscopy and nanoparticle tracking analysis for their morphological characteriza- tion. We employed GeLC-MS/MS combined with isotope-coded protein labeling to evaluate cell-derived proteins and LC-MS/MS label free spectral counting to quantify the EVs proteome. We found higher HER2 levels in both C5.2-derived EVs when compared with C5.2 cells, sug- gesting its preferential shuttling. Proteins capable of inducing malignant transformation are enriched in both C5.2 EV subsets, including two HER2-related proteins involved in cell motility and invasion, cofilin and CD44. MetaCore TM analysis indicated an enrichment of cell adhesion and cytoskeleton-remodeling pathways in C5.2 EVs, as well as proteins related to HER2 sig- naling, such as sphingosine-1-phosphate pathway. Together, our data indicate that in terms of protein content, distinct vesicle sets reinforce and complement each other. Our results also suggest that HER2-upregulated proteins from EVs may be relevant for cellular malignancy and can be potential biomarkers for HER2 + cancer patients. Keywords: Cell biology / Exosomes / Extracellular vesicles / HER2 / Microvesicles  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Extracellular vesicles (EVs) secreted by cells are important mediators of cell-cell communication [1]. EVs are diverse in terms of size and intracellular origin and their two most Correspondence: Dr. Diana Nunes, Laboratory of Medical Genomics, AC Camargo Cancer Center, Rua Tagu´ a 440, 1st floor, S˜ ao Paulo, SP 01508-010, Brazil E-mail: dianann@gmail.com Abbreviations: EV, extracellular vesicle; EX, exosome; ICPL, iso- topecoded protein label; MV, microvesicle; S1P, sphingosine-1- phosphate; RT, room temperature studied populations are generated through fundamentally different mechanisms of cellular vesiculation [2]: microvesi- cles (MVs) are formed through the outward blebbing of the plasma membrane, and exosomes (EXs) are derived from the inward budding of the plasma membrane in a process coor- dinated by the endosomal-sorting complex required for trans- port machinery [3]. MVs are between 0.1 and 1m in size and EXs are smaller (50—100 nm), but both have been shown to ∗ Additional corresponding authors: Dr. Daniel Martins-de-Souza and Dr. Emmanuel Dias-Neto, E-mail: danms90@gmail.com; emmanuel@cipe.accamargo.org.br Colour Online: See the article online to view Fig. 1 in colour. C  2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com