Sánchez-Bailón María Pilar, Calcabrini Annarica, Mayoral-Varo Víctor, Molinari Agnese, Wagner Kay-Uwe, Losada Jesús Pérez, Ciordia Sergio, Albar Juan Pablo, Martín-Pérez Jorge
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.
Oncotarget. 2015 May 30;6(15):13520-38. doi: 10.18632/oncotarget.3760.
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.
Src家族激酶（SFKs）参与肿瘤发生和转移。在此，我们通过四环素依赖性表达特异性shRNA-c-Src分析了c-Src在转移初始步骤中的作用，该shRNA可抑制转移性MDA-MB-231细胞中c-Src的mRNA和蛋白水平。c-Src的抑制并未改变细胞增殖或存活，但显著降低了非锚定依赖性生长。与Fak、小窝蛋白-1、桩蛋白和p130CAS的酪氨酸磷酸化/激活减少同时发生的是，c-Src的缺失也抑制了细胞迁移、侵袭和跨内皮迁移。对分泌蛋白组的定量蛋白质组学分析表明，在外泌体组分中检测到的Cyr61水平在shRNA-c-Src表达后降低。相比之下，细胞内Cyr61的表达未改变。Cyr61与顺式高尔基体gp74标记物和外泌体标记物CD63部分共定位，但c-Src的缺失并未改变它们在细胞内的分布。在SUM159PT细胞中，短暂抑制c-Src也降低了分泌的外泌体Cyr61水平。此外，在MDA-MB-231和SUM159PT中条件性表达c-Src显性负性突变体（SrcDN，c-Src-K295M/Y527F）也降低了分泌的Cyr61水平。在MDA-MB-231中短暂抑制Cyr61可抑制侵袭和跨内皮迁移。最后，在MDA-MB-231和SUM159PT中，一种中和性Cyr61抗体均抑制了迁移。总体而言，这些结果表明c-Src调节分泌蛋白，包括外泌体Cyr61，它们参与调节三阴性乳腺癌细胞的转移潜能。
