title: Cyr61 as mediator of Src signaling in triple negative breast cancer cells.
abstract: SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.
authors.affiliation: Departamento de Biología del Cáncer, Instituto de Investigaciones Biomédicas A. Sols (CSIC/UAM), Madrid 28029, Spain.
authors.forename: María Pilar
authors.initials: MP
authors.lastname: Sánchez-Bailón
authors.affiliation: Departamento de Biología del Cáncer, Instituto de Investigaciones Biomédicas A. Sols (CSIC/UAM), Madrid 28029, Spain.|Dipartimento Tecnologie e Salute, Istituto Superiore di Sanità, Roma 00161, Italy.
authors.forename: Annarica
authors.initials: A
authors.lastname: Calcabrini
authors.affiliation: Departamento de Biología del Cáncer, Instituto de Investigaciones Biomédicas A. Sols (CSIC/UAM), Madrid 28029, Spain.
authors.forename: Víctor
authors.initials: V
authors.lastname: Mayoral-Varo
authors.affiliation: Dipartimento Tecnologie e Salute, Istituto Superiore di Sanità, Roma 00161, Italy.
authors.forename: Agnese
authors.initials: A
authors.lastname: Molinari
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA.
authors.forename: Kay-Uwe
authors.initials: KU
authors.lastname: Wagner
authors.affiliation: Centro de Investigación del Cáncer (CSIC/USAL), Campus Unamuno, Salamanca 37007, Spain.
authors.forename: Jesús Pérez
authors.initials: JP
authors.lastname: Losada
authors.affiliation: Servicio de Proteómica, Centro Nacional de Biotecnología (CSIC), Madrid 28049, Spain.
authors.forename: Sergio
authors.initials: S
authors.lastname: Ciordia
authors.affiliation: Servicio de Proteómica, Centro Nacional de Biotecnología (CSIC), Madrid 28049, Spain.
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Albar
authors.affiliation: Departamento de Biología del Cáncer, Instituto de Investigaciones Biomédicas A. Sols (CSIC/UAM), Madrid 28029, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Martín-Pérez
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000081247:CSK Tyrosine-Protein Kinase; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D055515:Cysteine-Rich Protein 61 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D064726:Triple Negative Breast Neoplasms / Q000097:blood / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; C501848:CCN1 protein, human; D055515:Cysteine-Rich Protein 61; D012333:RNA, Messenger; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human
keywords: Cyr61; c-Src; invasion; migration; triple negative breast cancer