c-Src signaling in triple negative breast cancer cells: role of Cyr61

Abstract

The SFKs (Src Family Kinases) control cellular pathways involved in division, motility, adhesion, angiogenesis, and survival. Therefore, their deregulation is associated with tumorigenesis, and metastasis. c-Src is overexpressed and/or aberrantly activated in epithelial tumors: pancreatic, colorectal, prostatic, ovarian, breast, etc. We previously showed that SFKs catalytic inhibitors (Dasatinib, PP2, and SU6656) reduce proliferation, migration, and invasiveness of MDA-MB-231. Here, we analyzed c-Src contribution to initial steps of metastasis by Tet-On conditional expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in MDA-MB-231. 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 inhibited migration, invasion, transendothelial migration, and reduced MMP2, MMP7 and MMP9 in secretome. Quantitative proteomic analyses of secretome showed that Cyr61 levels, detected in exosomal fraction, were diminished upon shRNA-c-Src expression. However, 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 distribution. In SUM159PT, transient c-Src suppression also reduced secreted exosomal Cyr61. Furthermore, conditional expression of c-Src dominant negative mutant (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 exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.