mda-9/Syntenin promotes metastasis in human melanoma cells by activating c-Src

Abstract

The scaffold PDZ-domain containing protein mda-9/syntenin functions as a positive regulator of cancer cell progression in human melanoma and other tumors. mda-9/Syntenin regulates cell motility and invasion by altering defined biochemical and signaling pathways, including focal adhesion kinase (FAK), p38 mitogen-activated protein kinase (MAPK) and NF-kappaB, but precisely how mda-9/syntenin organizes these multiprotein signaling complexes is not well understood. Using a clinically relevant human melanoma model, we demonstrate that mda-9/syntenin physically interacts with c-Src and this communication correlates with an increase in FAK/c-Src complex formation and c-Src activation. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin or addition of c-Src siRNA, suppresses melanoma cell migration, anchorage-independent growth, and spontaneous tumor cell dissemination in vivo in a human melanoma animal metastasis model. These data are compatible with a model wherein interaction of MDA-9/syntenin with c-Src promotes the formation of an active FAK/c-Src signaling complex, leading to enhanced tumor cell invasion and metastatic spread. These provocative findings highlight mda-9/syntenin and its interacting partners as promising therapeutic targets for intervention of metastasis.

Fig. 1.

Expression of mda-9/syntenin correlates with an increase in c-Src/FAK complex formation in human melanoma cell lines derived from tumors at different stages of progression. (A) Serum-starved cells were plated on fibronectin and analyzed by Western blot with anti-c-Src antibody or phosphospecific antibody to c-Src Tyr⁴¹⁶. (B) Cell lysates were immunoprecipitated with anti-c-Src antibodies followed by Western blotting with anti-FAK-Tyr³⁹⁷, anti-FAK antibodies, anti-c-Src Tyr⁴¹⁶, or anti-c-Src antibodies. (C) Lysates of infected cells with the indicated adenovirus were analyzed by Western blotting for c-Src activity. CTR and Ad.null vector refer to untreated control cells and adenovirus vector not expressing mda-9/syntenin, respectively.

Fig. 2.

Pharmacological inhibition of c-Src or c-Src siRNA reduces mda-9/syntenin-induced c-Src activation and FAK phosphorylation at Tyr³⁹⁷. Serum-starved cells either uninfected or infected with Ad.mda-9/S (50 pfu/cell) were plated on fibronectin and pretreated with DMSO (0), the active Src inhibitor PP2, or its inactive analog PP3 (A and B) or transfected with c-Src siRNAs (10 or 50 nM) or control nonspecific siRNAs (C and D). Lysates were analyzed by Western blotting with the indicated phosphotyrosine antibodies. Ctr refers to scrambled-siRNA or siRNA to GAPDH.

Fig. 3.

MDA-9/syntenin interacts with c-Src. (A) Fluorescent confocal micrographs of the metastatic variant T1P26 plated on fibronectin showing immunolocalization of phopho-c-Src protein and MDA-9/syntenin (arrows). Serum-starved M4Beu. cells constitutively expressing HA-tagged wt or HA-tagged Mda-9/syntenin mutant or serum-starved cells either uninfected or infected with Ad.null or Ad.mda-9/AS (100 pfu/cell) were plated on fibronectin and immunoprecipitated with anti-MDA-9/syntenin antibodies followed by Western blotting with anti-c-Src or anti-MDA-9/syntenin antibodies (B) or anti-c-Src antibodies followed by anti-MDA-9/syntenin or anti-c-Src antibodies (C) or anti-c-Src antibodies followed by anti-HA-MDA-9/syntenin or anti-c-Src antibodies (D).

Fig. 4.

Silencing of endogenous c-Src by siRNAs inhibits mda-9/syntenin-induced anchorage-independent growth, increased cell migration, and spontaneous melanoma metastasis in vivo. Anchorage-independent growth (A) and migration across matrigel-coated filters (B) of serum-starved M4Beu. cells constitutively expressing wild type or the mda-9/syntenin double PDZ deleted mutant or serum-starved cells infected with Ad.null or Ad.mda-9/S and transfected with control or C-Src siRNA. Spontaneous lung metastases (C) with cells infected with Ad.null (7GP, T1P26) or Ad.mda-9/S (FM516-SV, M4Beu.) and transfected with jetPEI-complexed C-Src siRNAs or a PEI-complexed unrelated siRNA administred intratumorally on days 2, 4, 6, 10, 14, and 18 after tumor inoculation, or serum-starved M4Beu. Constitutively expressing wild type or the mda-9/syntenin double PDZ deleted mutant. Representative photomicrographs of anchorage-independent growth (A), cell migration (B), and lungs of control and treated animals (C) shown on right. The mean ± SD of metastatic lung nodules was determined using a dissecting microscope to examine excised/fixed/stained lungs.

Fig. 5.

Hypothetical model of signal transduction pathways coordinately regulated by MDA-9/syntenin through its interaction with c-Src. MDA-9/syntenin interaction with c-Src results in clustering of c-Src/FAK signaling complexes at high concentrations on the plasma membrane. The activated c-Src/FAK complexes activate the p38 MAPK/NF-κB pathways that regulate expression of genes involved in migration and invasion and thus play a crucial role in MDA-9/syntenin-mediated tumor progression.