Fig. 6

SRGN promotes cell migration through Src-elicited focal adhesion turnover. a After serum-starvation for 24 h, H1299/Mock and H1299/SRGN cells were plated in FN-coated dishes. In 30 min, cells were lysed and assayed for Src phosphorylation. In parallel, serum-starved cells were subjected to western blot analysis of phosphorylated paxillin and ERK/MAPK, and co-immunoprecipitation assay for paxillin/FAK adhesion complex formation. b H460/sh-CTRL and H460/sh-SRGN cells were cultured in serum-free medium for 24 h and examined for Src phosphorylation and paxillin/FAK complex formation. c H1299/SRGN cells were cultured in serum-free medium for 24 h, treated with PP2 (10 μM) or DMSO for 3 h, and phosphorylation of Src and paxillin, and paxillin/FAK complex formation were assessed. d Src expression was depleted in H1299/SRGN cells by shRNA-mediated knockdown approach, and phosphorylation of paxillin and the formation of paxillin/FAK complex were assessed in the control-knockdown (sh-CTRL) and Src-knockdown (sh-Src) cells. e-f H1299/Mock and H1299/SRGN cells (e) or H460/sh-CTRL and H460/sh-SRGN cells (f) were treated with or without PP2, and subjected to Boyden chamber migration assay. Migrated cells were counted in 3 h. Western blot analysis of phosphorylated Src and total Src was shown. Data are presented as the mean ± SD of three independent experiments. ***P < 0.001 by Student’s t-test. g HuTu80/CD44 cells were incubated with CM harvested from H1299/Mock and H1299/SRGN cells that had been treated with or without ChaseABC for 24 h, followed by western blot analysis of designated proteins. h H1299/Mock, H1299/SRGN and H1299/SRGN(S/A) cells were cultured in serum-free medium for 24 h, and subjected to western blot analysis of phosphorylation of Src, paxillin and other proteins as indicated. i A working model is proposed for SRGN-mediated cell migration. SRGN in the tumor microenvironment binds to tumor cell surface CD44 via its CS-GAGs, and promotes Src activation and subsequent paxillin phosphorylation and the dissociation of paxillin/FAK adhesion complex, to accelerate focal adhesion turnover. SRGN also promotes the activation of Rac1 and Cdc42 to enhance cytoskeleton reorganization, leading to increased cell migration