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-κB, 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.

A devastating outcome of cancer progression is metastasis. Understanding and preventing this process and its dire consequences remain formidable obstacles in achieving successful treatment outcomes in advanced cancer patients. In the context of advanced melanoma, the response rate of patients with metastatic disease to single agent chemotherapy or combination therapies is frequently less than 10% (1). Given this bleak picture, it is vital to develop new, efficacious, and rationally designed treatment strategies to, ideally, prevent or effectively treat metastasis. In this context, defining relevant genes that are seminal regulators of metastasis provide an entry point for developing improved therapies.

Melanoma differentiation associated gene-9 (mda-9) was cloned in our laboratory (2, 3) as a unique gene displaying biphasic expression during terminal differentiation of human melanoma cells treated with a combination of fibroblast IFN (IFN-β) and the antileukemic compound mezerein (MEZ). These changes are consistent with early enhancement followed by decreased expression during the course of reversion of the cancer phenotype in melanoma cells (2, 3). mda-9, also called syntenin, expression displays an inverse relationship with tumor progression, with low-level expression in melanocytes and radial growth phase (RGP) primary melanoma versus elevated expression in vertical growth phase (VGP) primary melanoma and metastatic melanomas (4, 5). The level of mda-9/syntenin is also elevated in multiple additional cancers, including breast and gastric carcinomas, suggesting an expanded involvement in tumor progression (6). Recently, we confirmed the importance of mda-9/syntenin in metastasis in vivo, providing direct support for mda-9/syntenin as an essential gene controlling melanoma progression and metastasis (4).

mda-9/Syntenin is an evolutionary conserved cytosolic protein representing a unique member of an expanding family of scaffolding proteins with highly potent and diverse biological activities (7, 8). A notable feature of mda-9/syntenin is the presence of tandem PDZ domains of 83 and 80 amino acid residues, respectively (PDZ1 and PDZ2), that are required for the assembly and organization of diverse cell signaling processes occurring at the plasma membrane (7, 8). On a structural level, mda-9/syntenin has remarkable flexibility due to its ability to specifically bind to internal or C-terminal sequences of target proteins (7, 8). This plasticity allows MDA-9/syntenin to participate in multiple biological functions, including receptor clustering, protein trafficking, activation of the transcription factor Sox4, syndecan recycling through endosomal compartments, and cell adhesion (7, 8).

An intriguing property of mda-9/syntenin is its ability to induce morphological changes in cell shape in multiple cancers, including melanoma (4, 6). We previously documented that such changes orchestrated by the actin cytoskeleton lead to an increase in anchorage-independent growth and invasiveness of melanoma cells in a FAK-dependent manner that involve activation of key signaling molecules, including p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) (4). Recent studies demonstrate that mda-9/syntenin induces NF-κB activation, representing a potential mechanism by which mda-9/syntenin acting through FAK promotes cancer progression to metastasis in an MMP-2-dependent manner (9). However, it is unclear precisely how mda-9/syntenin holds multiprotein signaling complexes together or how these complexes impact the cells' migration machinery.

c-Src, a prototype of the nine-member family of structurally related Src family tyrosine kinases (SFKs), is implicated in various biological processes associated with cytoskeletal organization, including increased cell motility, invasiveness, and survival (10). Upon integrin engagement, FAK together with Src form a dual-kinase complex that generates signals important for regulation of the migration machinery that promote tumor growth and metastasis (11, 12). Additionally, several studies implicate c-Src in the pathogenesis and progression of multiple cancers, including breast, pancreatic, prostate, and melanoma, emphasizing the importance of c-Src in cancer biology (12–15).

Using a clinically germane human melanoma model, we presently establish a mechanism for c-Src activation by mda-9/syntenin. mda-9/Syntenin physically interacts with c-Src, and this interaction correlates with an increase in the formation of an active FAK/c-Src signaling complex that ultimately promotes cell motility, extracellular matrix (ECM) invasion, and melanoma metastasis formation in vivo. These original observations support the hypothesis that mda-9/syntenin may represent a promising new molecular target for melanoma therapy.

Results

High-Constitutive pp60^c-src Activation Is Observed in Melanoma Cells Overexpressing mda-9/Syntenin.

mda-9/Syntenin expression is elevated in metastatic melanoma cells versus normal melanocytes, promoting cell motility toward fibronectin and invasion by enhancing FAK activity (4). Because SFKs are required for specific aspects of tumor progression, including cell motility and invasion (10, 11), we investigated a possible relationship between SFKs and mda-9/syntenin in melanoma cells with differing phenotypes. When seeded on polyL-lysine-coated plates, normal immortal melanocytes (FM516-SV) and poorly metastatic melanoma cells (M4Beu.), which expressed low levels of mda-9/syntenin, contained very low or undetectable levels of phosphorylated pp60^c-src at pY418 (Fig. 1A). The level of phosphorylation at pY418 was modestly induced in both FM516-SV and M4Beu. cells when plated on fibronectin in serum-free media. In contrast, plating metastatic cells (which express high levels of mda-9/syntenin), including metastatic melanoma variants, T1P26 and 7GP, as well as cell lines derived from patients with metastatic melanomas, MeWo and HO-1, onto fibronectin induced a robust increase in pp60^c-src phosphorylation compared with either parallel cultures plated on polyL-lysine or FM516-SV or M4Beu. cells plated on fibronectin.

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.

Because FAK association with c-Src corresponds with an active signaling complex (16), c-Src association with FAK was compared in lysates of poorly and highly metastatic cells. As expected, coimmunoprecipitation of c-Src with FAK was significantly elevated in T1P26 and 7GP cells versus FM516-SV and M4Beu. cells (Fig. 1B). In addition, the levels of a molecular complex containing both FAK and Src was greater in highly metastatic cells than in weakly metastatic cells (Fig. 1B). Moreover, the c-Src that associated with FAK in highly metastatic cells, as detected with anti-pY418 antibody, showed greater phosphorylation than in weakly metastatic cells.

Forced Expression or Inhibition of Expression of mda-9/Syntenin by Adenovirus Transduction Regulates src Activity in Human Melanoma Cells.

We next manipulated the expression levels of mda-9/syntenin in FM516-SV and M4Beu., with low metastatic potential, and T1P26 and 7GP, which generate a high frequency of spontaneous metastasis in immunosuppressed neonatal rats (9, 17, 18). As evident in Fig. 1C, infection of normal FM516-SV immortal melanocytes or M4Beu. cells with 100 pfu/cell of Ad.mda-9/S had greater src phosphorylation at Tyr⁴¹⁸ (∼3-fold increase) upon adhesion to fibronectin than cells infected with the same multiplicity of infection of Ad.null (virus lacking the mda-9/syntenin gene). In contrast, Ad.mda-9/AS-infected T1P26 or 7GP cells displayed a significant reduction in src phosphorylation (∼3-fold reduction) when compared with uninfected control or Ad.null-infected cells.

Silencing of Endogenous c-Src by siRNA Inhibits mda-9/Syntenin-Mediated Activation of the FAK/Src Pathway.

To evaluate the role of pp60^c-src kinase in mda-9/syntenin-mediated phosphorylation of FAK at Tyr³⁹⁷, we initially used PP2, a selective pharmacological antagonist of SFKs. As evident in Fig. 2A, after infection of FM516-SV or M4Beu. cells with Ad.mda-9/S (100 pfu/cell) and plating on fibronectin in serum-free medium, a dose-dependent decrease in the phosphorylation of FAK at Tyr³⁹⁷ was observed with PP-2 (1–10 μM), in comparison with the inactive congener PP-3. Similarly, consistent with a role of SFKs, PP-2 induced a dose-dependent decrease of FAK phosphorylation at Tyr³⁹⁷ in T1P26 and 7GP cells in comparison with PP-3 (Fig. 2B). Blocking Src kinases pharmacologically in additional metastatic human melanoma cell lines, including SH-1, 3S5, C8161, FO-1, and HO-1, resulted in a similar reduction in FAK phosphorylation at the Tyr³⁹⁷ residue (data not shown). These results support a role of Src or other SFKs in mda-9/syntenin-induced FAK activation.

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.

To further confirm an association between mda-9/syntenin expression and src and FAK phosphorylation, we used siRNA designed to specifically knockdown human c-Src. As apparent in Fig. 2C, total c-Src protein was significantly reduced by 10 nM siRNA complexes in Ad.mda-9/S-infected FM516-SV or M4Beu. cells (50 pfu/cell) plated in serum-free medium on fibronectin, and a concentration of 50 nM reduced c-Src protein to nearly undetectable levels. Most importantly, knockdown of endogenous c-Src in FM516-SV and M4Beu. cells was accompanied by a dose-dependent decrease of c-Src and FAK phosphorylation (∼3-fold and ∼2.5-fold reduction, respectively) when compared with 50 nM of nontargeting siRNA complexes or siRNA targeting GAPDH (Fig. 2C). Similarly, treatment of melanoma metastatic variants, T1P26 and 7GP cells, with 10 and 50 nM siRNA complexes resulted in a significant dose-dependent decrease in total c-Src protein (Fig. 2D). As expected, although knockdown of the c-Src gene did not change total FAK levels in T1P26 and 7GP cells, it did reduce in a dose-dependent manner the level of active c-Src and FAK (Fig. 2D). The specificity of c-Src knockdown was confirmed by the fact that the level of EF1α protein levels remained unchanged by the on-target c-Src siRNAs (data not shown). In total, these findings employing pharmacological and genetic approaches to block c-Src support the hypothesis that mda-9/syntenin plays a critical role in the activation of the Src/FAK signaling pathway in this melanoma model.

mda-9/Syntenin Physically Interacts with c-Src, Which Correlates with an Increase in FAK/Src Complex Formation in Human Melanoma Cells.

MDA-9/syntenin colocalizes with FAK upon adhesion of melanoma cells to fibronectin and Ad.mda-9/S infection enhances phosphorylation of FAK at Tyr³⁹⁷ (4). Ad.mda-9/S infection also stimulates pp60^c-src kinase activity in melanoma cells, and expression of mda-9/syntenin correlates with an increase in FAK/c-Src complex formation in melanoma cells (Fig. 1 A and B). These findings prompted us to investigate whether MDA-9/syntenin colocalizes with c-Src. Merging of the immunofluorescent images for MDA-9/syntenin and c-Src showed that MDA-9/syntenin significantly colocalized with c-Src in T1P26 melanoma cells plated on fibronectin, predominantly in areas that correspond to peripheral focal adhesion, whereas other fractions localized in a diffuse manner in the cytoplasm and nucleus (Fig. 3A). In addition, both proteins colocalize in discrete structures in specific areas in the periphery of the cell, indicating that these focal adhesions may differ in terms of their protein content and partners that are mediated by the PDZ domains of MDA-9/syntenin. Of potential interest, both proteins also colocalize in or near the cell nucleus, suggesting that these two proteins could interact and may promote transcriptional activities (7, 8, 19).

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).

Experiments were next performed to determine whether MDA-9/syntenin and c-Src could associate with FAK in vivo. As anticipated, coimmunoprecipitation of c-Src with MDA-9/syntenin was very robust in metastatic melanoma variants T1P26 and 7GP plated on fibronectin, but was significantly reduced in FM516-SV and weakly metastatic M4Beu. melanoma cells (Fig. 3B). Reprobing the membrane with anti-MDA-9/syntenin antibody confirmed reduced expression in FM516-SV and M4Beu. cells, with significant elevated expression in T1P26 and 7GP cells. These findings indicate that, in T1P26 and 7GP cells, MDA-9/syntenin forms a complex with c-Src that may be a critical step in the acquisition of an aggressive phenotype by evolving melanoma cells.

An antisense strategy also confirmed the physical interaction between MDA-9/syntenin and c-Src. As shown in Fig. 3C, a significant level of MDA-9/syntenin was coimmunoprecipitated with anti-c-Src antibody in Ad.null-infected cells. In contrast, immunoprecipitation with anti-c-Src antibody did not pull down MDA-9/syntenin after infection of metastatic melanoma cells with Ad.mda-9/AS (50 pfu/cell). Additionally, deletion of the two PDZ domains of MDA-9/syntenin prevented interaction with c-Src, providing further documentation of the in vivo interaction of MDA-9/syntenin with c-Src (Fig. 3D).

Anchorage-Independent Growth, Cell Migration, and Metastasis of Melanoma Cells in Vivo Depend on mda-9/Syntenin-Mediated FAK/Src Activation.

Given the central role the c-Src/FAK signaling pathway plays in regulating cell motility in cancer cells (11, 20, 21), we addressed the functional consequence of on-target and off-target siRNAs on mda-9/syntenin-induced FAK/c-Src complex formation in vivo. We first assessed whether melanoma cell lines require c-Src to support anchorage-independent growth using a soft agar assay that distinguishes between tumorigenic cells with the capacity to metastasize in vivo versus tumorigenic cells that lack this ability (4, 22). As demonstrated in Fig. 4A, mda-9/syntenin overexpression in FM516-SV and M4Beu. cells resulted in more and larger colonies growing in soft agar than untreated control or Ad.vec-infected cells. In contrast, deletion of the two MDA-9/syntenin PDZ domains or reduction of c-Src protein by specific siRNAs significantly suppressed (∼85%–90% reduction) the mda-9/syntenin-induced increase in anchorage-independent growth compared with wild-type MDA-9/syntenin or nontargeting siRNA control complexes (Fig. 4A). Similarly, transfection of T1P26 and 7GP cells with c-Src siRNAs reduced by more than 85% the ability of these two highly metastatic cell lines to form soft agar colonies compared with nonspecific control siRNA transfected cells (Fig. 4A), supporting a potential involvement of mda-9/syntenin in the activation of c-Src/FAK complexes in this process.

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.

The requirement for c-Src in maintaining the transformed phenotype was further investigated by cell invasion/migration assays. As evident in Fig. 4B, overexpression of mda-9/syntenin significantly enhanced the invasive ability of FM516-SV and M4Beu. cells, with greater effects observed in M4Beu.cells than in FM516-SV cells (4). This increased invasion following infection with Ad.mda-9/S was inhibited by more than 85% to 90% with the deletion construct of MDA-9/syntenin lacking the PDZ tandem domains or c-Src siRNAs compared with wild-type MDA-9/syntenin or control nonspecific siRNAs (Fig. 4B). Similarly, transfection of T1P26 and 7GP cells with c-Src siRNAs significantly reduced (∼85%–90%) the invasive ability of the two highly metastatic cell lines compared with control (Ctr) nonspecific siRNAs (Fig. 4B), further establishing an involvement of the Src/FAK signaling pathway in mda-9/syntenin-enhanced migration and invasion.

To confirm a direct involvement of mda-9/syntenin-induced activation of the c-Src/FAK signal transduction pathway in tumor cell progression, we analyzed the effect of c-Src siRNA complexes in a spontaneous metastasis model. We reasoned that if this mode of regulation operates in living cells, we would expect that a reduction in endogenous c-Src would decrease c-Src levels, resulting in a decrease in melanoma growth and mestastasis. Because of the transient nature of siRNA knockdown and the technical challenges of tail vein injections in newborn rats, multiple siRNA treatments were given intratumorally on days 2, 4, 6, 10, 14, and 18. As shown in Fig. 4C, the number of metastatic lung lesions was significantly enhanced in animals receiving Ad.mda-9/S-infected FM516-SV or M4Beu. cells (50 pfu/cell), with a more dramatic enhancement observed in M4Beu. cells (75 ± 12 lung nodules vs. 25 ± 7 in Ad.null-infected cells) than in FM516-SV cells. In contrast, multiple subcutaneous injections of c-Src siRNAs from day 2 to day 18 resulted in a significant decrease in the average number of metastatic surface tumor nodules per lung lobe (5 ± 3 lung nodules in M4Beu. cells treated with active siRNAs vs. 73 ± 10 in cells treated with control siRNA), with no change in the rate of primary tumor growth (data not shown). Similarly, deletion of the two MDA-9/syntenin PDZ domains significantly reduced lung metastases (4 ± 2 lung nodules in M4Beu. cells transfected with mutant vs. 65 ± 7 in cells transfected with wild type MDA-9/syntenin). Although treatment of mice receiving Ad.mda-9/S-infected FM516-SV cells with active siRNAs resulted in a less dramatic decrease in lung metastases than that observed in M4Beu. cells, the difference was still evident (Fig. 4C). In contrast, silencing c-Src in highly metastatic variants T1P26 and 7GP through multiple treatments with active siRNAs resulted in a significant decrease in the average number of metastatic surface tumor nodules per lung lobe (120 ± 13 and 115 ± 14 lung nodules in T1P26 and 7GP cells, respectively, treated with control nonspecific siRNAs vs. 4 ± 2 and 3 ± 1 in cells, respectively, treated with active siRNAs), approaching that observed in Ad.mda-9/S-infected M4Beu. cells treated with c-Src siRNAs. Upon further microscopic inspection of the lungs, no micrometastases were found in any animal among a group of 10 mice after 3 weeks (incidence of metastasis P < 0.002 vs. control). Extending the incubation time to >21 days resulted in small visible nodules growing at a slower rate than controls in only one of 10 mice. These noteworthy in vivo studies confirm a cause-and-effect relationship between mda-9/syntenin-induced Src activation and metastatic competence in human melanoma cells.

Discussion

Prior studies confirm that mda-9/syntenin stimulates motility through pathways involving FAK, p38 MAPK, and NF-κB, leading to secretion of MMP-2 (4, 9). However, despite these intriguing observations, it is not fully understood how mda-9/syntenin orchestrates these signaling molecules to enhance cancer cell motility and metastasis. A complex network of protein-protein interactions characterizes the structural organization of focal adhesions, involving known signaling molecules that play functional roles in various cellular activities and other less well-defined pathways (23, 24). We presently show that mda-9/syntenin interacts with c-Src through its PDZ domain and activates the c-Src/FAK signaling pathway to maximize tumor cell motility and anchorage-independent growth of melanoma cells. mda-9/Syntenin levels directly correlate with increased c-Src activity in a human melanoma model that closely mimics the early events of metastasis in humans. Additionally, down-regulation of c-Src expression by RNA interference or deletion of the two PDZ domains of mda-9/syntenin inhibited spontaneous pulmonary metastases of melanoma cells in vivo, implying a functional cooperation between mda-9/syntenin and c-Src expression during tumor progression and melanoma metastasis.

These findings raise intriguing questions as to how mda-9/syntenin activates c-Src and how it then regulates increased c-Src/FAK complex formation. Cell migration is a dynamic multistep process regulated by autophosphorylation of FAK, which synergizes with SFKs to further recruit and activate defined downstream effectors (25). A critical event in integrin-mediated FAK signaling is phosphorylation of Tyr³⁹⁷ (11, 25). Notably, pY397FAK creates a high-affinity binding site for SH2 domains of SFKs, and this interaction promotes Src kinase activity through a conformational change. Activated c-Src bound to pY397FAK then phosphorylates additional sites on FAK, which promote the assembly of distinct higher-order individual signaling complexes, thereby providing a mechanism for coordinating signaling through multiple pathways (24, 25). Normally, inactive c-Src exists as a tight complex in which the SH2 domain interacts with phosphotyrosine at position 527 localized in the C-terminal region of the protein. Src activation results in the displacement of C termini from the SH2 domain proteins, promoting interactions with SH2 and/or SH3 ligands that prime c-Src for activation and thereby allowing phosphorylation of the kinase on Tyr⁴¹⁶ (26, 27).

c-Src and FAK are already constitutively activated in normal immortal melanocytes and in poorly metastatic melanoma cells as shown in this and previous studies (4). Theoretically, binding of MDA-9/syntenin to c-Src through its PDZ domain-binding motifs may enable MDA-9/syntenin to assemble large c-Src-FAK complexes, which greatly enhance activation of the c-Src-FAK complex, leading to amplification of p38/NF-κB signaling pathways (7–9). In this study, we observed that stimulation of integrin signaling with fibronectin, which recruits the SH3 domain of c-Src to the cytoplasmic domain of integrin and eventually leads to c-Src activation (28), promoted the formation of an active c-Src-FAK signaling complex in melanoma cells overexpressing mda-9/syntenin from the small pool of c-Src proteins with which FAK was associated (Fig. 1B). This increase was attenuated by treatment with PP2 or siRNA knockdown of c-Src, suggesting that c-Src bound to MDA-9/syntenin functionally cooperates with FAK-induced activation of the p38 MAPK/NF-κB signaling pathways to promote migration of melanoma cells (9). The functional cooperation between MDA-9/syntenin and c-Src in human cancer is supported by a number of reports showing that cancer progression in multiple cancers is associated with enhanced expression of mda-9/syntenin in which c-Src activity is frequently elevated (4–8, 12). In these contexts, our present studies support a central role for mda-9/syntenin in the c-Src-FAK axis that regulates migration/invasion of melanoma cells (29). Analysis of the double PDZ deletion mutant argues that engagement of the PDZ domains is essential for interactions between MDA-9/syntenin and c-Src that in turn promote cancer development and progression. Signaling molecules that have PDZ domains are known to organize proteins in the same signaling cascade to form macromolecular signaling complexes (7, 8). Although MDA-9/syntenin has only two PDZ domains, it can form complexes with itself, which may allow other cytoskeletal proteins at the focal contacts, including FAK, to be assembled in combination with c-Src (7, 8). A previous study reported that formation of clustered NMDA (N-methyl-D-aspartate) receptors by a neuronal membrane-associated guanylate kinase (MAGUK) PDZ domain, called PSD-95, precedes c-Src-mediated receptor phosphorylation (30). Consequently, loss of mda-9/syntenin expression would result in a loss of its ability to interact with sufficient efficiency with c-Src to promote signaling, and would therefore have a profound effect on c-Src-FAK signaling pathways. Further structural and biochemical studies are needed to fully understand the basis for specificity of the MDA-9/syntenin/c-Src interactions.

In summary, we have now uncovered a new role of mda-9/syntenin as an important PDZ scaffolding protein that enhances the assembly of stable c-Src-FAK signaling complexes, initiating a signaling cascade that results in enhanced anchorage independence, cell motility, and metastatic competence in human melanoma cells. Our findings support a hypothetical model in which MDA-9/syntenin through its interaction with itself and with c-Src enables c-Src/FAK signaling complexes clustered at high concentrations on the plasma membrane to amplify signaling through FAK intermolecular autophosphorylation (Fig. 5). The activated c-Src-FAK complexes may then signal through the p38 MAPK/NF-κB signaling pathways, leading to enhanced cell motility, invasion, and metastasis as shown in this and previous studies (4, 9, 31). The present findings suggest that MDA-9/syntenin and its interacting partners represent potential therapeutic targets to reduce cancer metastasis. Targeted therapies that inactivate MDA-9/syntenin/c-Src complexes via antisense, siRNA, or small molecule inhibitors may provide a rational molecular target-based approach to improve the prognosis of patients with metastatic disease.

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.

Materials and Methods

Reagents and Cell Lines.

Antibodies for Western blotting, immunoprecipitation, and immunochemistry included phosphoantibodies, anti-c-Src pY418 and anti-FAK pY397 (Biosource International), anti-c-Src, (Santa Cruz Biotechnology), anti-FAK (Transduction Laboratories), and anti-MDA-9/syntenin polyclonal antibody (Alpha-Diagnostic International). PP2 and PP3 inhibitors were obtained from Sigma-Aldrich. Normal immortal human melanocyte (FM516-SV), M4Beu. poorly metastatic human melanoma cell line, highly metastatic variants T1P26 and 7GP derived from M4Beu. parental cells, and metastatic melanoma cell lines MeWo and HO-1 were cultured as described (4).

Virus Construction, Purification, and Infectivity Assays.

Construction and characterization of Ad.mda-9/S and Ad.mda-9/AS were performed as described (4). Cells were infected with 50–100 plaque-forming units/cell of the indicated adenovirus, then serum starved and plated on fibronectin as described previously (4).

Plasmid Construction and Transfection Assays.

M4Beu. cells were cotransfected with pTK.Hygr and wild-type HA-MDA-9/syntenin or a mutant construct lacking PDZ-1 and PDZ-2 domains (generously provided by Dr. P.J. Coffer, Departments of Immunology and Pediatrics, University Medical Center, Utrecht, The Netherlands), using LipofecTAMINE 2000. Forty hours after transfection, standard methods were used to generate stable cell lines.

c-Src Silencing by RNA Interference.

A pool of two targeted sequences that mediate the silencing of c-Src expression were prepared using the Silencer siRNA kit (Ambion). The targeted sequences were: 5′-AACAAGAGCAAGCCCAAGGAT-3′ (52–71 bp) and 5′-AAGCACUACAAGAUCCGCAAG-3′ (607–628 bp). Twelve hours postinfection, cells were transfected with c-Src siRNAs or an irrelevant RNA duplex using siPORTAmine transfection reagent as recommended by the manufacturer.

Immunoprecipitation and Western Blotting Analysis.

Cell extracts in RIPA buffer were prepared, and equal amounts of proteins were resolved in SDS/PAGE, transferred, and evaluated for c-Src and FAK expression and activity with the indicated antibodies as previously described (4). For coimmunoprecipitations, equivalent amounts of cell lysates were incubated for 2 h at 4°C with antibodies to c-Src coupled to protein G-Sepharose as described previously (32). Immunoprecipitates were extensively washed, and the eluted precipitates were resolved by SDS/7% PAGE, transferred, and probed with the appropriate antibodies.

Restrictive Anchorage-Independent Growth, Invasion/Migration, and Metastasis Assays.

Serum-starved cells (2 × 10⁵) either uninfected or infected with the indicated adenovirus were transfected 12 h later with control or c-Src siRNAs by using LipofectAMINE 2000 transfection reagent. After 2 days, the cells were trypsinized and plated in 6 cm dishes. Colonies of >50 cells were scored, and colony formation in soft agar was determined as described (4, 22). Invasion/migration assays were performed as described (4). The spontaneous metastatic ability was determined by inoculating 10⁶ cells subcutaneously into the abdomen of immunosuppressed Wistar rats as described (4, 18). Three weeks later, lung invasion was evaluated by counting pulmonary nodules under a dissecting microscope. The siRNAs were mixed with JetPEI^TM transfection reagent following the manufacturer's instructions (Polyplus Transfection) and injected intratumorally in mice as indicated.