Abstract
Members of the Src family kinases (SFK) can modulate diverse cellular processes, including division, death and survival, but their role in autophagy has been minimally explored. Here, we investigated the roles of Lyn, a SFK, in promoting the survival of human glioblastoma tumor (GBM) cells in vitro and in vivo using lentiviral vector-mediated expression of constitutively-active Lyn (CA-Lyn) or dominant-negative Lyn (DN-Lyn). Expression of either CA-Lyn or DN-Lyn had no effect on the survival of U87 GBM cells grown under nutrient-rich conditions. In contrast, under nutrient-deprived conditions (absence of supplementation with L-glutamine, which is essential for growth of GBM cells, and FBS) CA-Lyn expression enhanced survival and promoted autophagy as well as inhibiting cell death and promoting proliferation. Expression of DN-Lyn promoted cell death. In the nutrient-deprived GBM cells, CA-Lyn expression enhanced AMPK activity and reduced the levels of pS6 kinase whereas DN-Lyn enhanced the levels of pS6 kinase. Similar results were obtained in vitro using another cultured GBM cell line and primary glioma stem cells. On propagation of the transduced GBM cells in the brains of nude mice, the CA-Lyn xenografts formed larger tumors than control cells and autophagosomes were detectable in the tumor cells. The DN-Lyn xenografts formed smaller tumors and contained more apoptotic cells. Our findings suggest that on nutrient deprivation in vitro Lyn acts to enhance the survival of GBM cells by promoting autophagy and proliferation as well as inhibiting cell death, and Lyn promotes the same effects in vivo in xenograft tumors. As the levels of Lyn protein or its activity are elevated in several cancers these findings may be of broad relevance to cancer biology.
Highlights
Lyn is one of eight members of the Src family of kinases (SFK) expressed in human cells [1]
The levels of Lyn activity were manipulated by transducing U87 glioblastoma tumor (GBM) cells with lentiviral vectors expressing a constitutivelyactive Lyn (CA-Lyn) construct or a dominant-negative Lyn (DN-Lyn) construct
After expressing the constructs in U87 GBM cells using lentiviral vector, stable populations of cells were selected with puromycin, sorted for green fluorescent protein (GFP) expression to maintain homogeneous, expression-positive populations
Summary
Lyn is one of eight members of the Src family of kinases (SFK) expressed in human cells [1]. The functions of SFKs appear to be influenced by the microenvironment as well as cell type and post-translational modifications [4,6,7,8,9], little attention has been paid to the role of SFKs in promoting cell survival through regulation of autophagy. A potential role for SFKs in autophagy is suggested by the reports that Dasatinib, which inhibits multiple SFKs as well as Bcr-Abl, induces autophagy in multiple types of cancer cells, including GBM, under nutrient-rich conditions [10,11]. C-Src has been shown to localize to autophagosomes in focal adhesion kinase (FAK)-deficient cells under nutrient-rich conditions [12]
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