Aberrant adhesion signaling pathways in cancer cells underlie their deadly invasive capabilities. The adhesion-related PDZ adapter protein mda-9/syntenin is a positive regulator of cancer cell progression in breast cancer, melanoma, and other human cancers. In this study, we report that mda-9/syntenin mediates adhesion-mediated activation of protein kinase Cα (PKCα) and focal adhesion kinase (FAK) by fibronectin (FN) in human breast cancer and melanoma cells. FN rapidly stimulated the expression of mda-9/syntenin and the activation of PKCα prior to activation of FAK. Inhibiting PKCα suppressed basal or FN-induced expression of mda-9/syntenin, as well as cell migration and invasion toward FN stimulated by mda-9/syntenin. Several lines of evidence suggested that activation of PKCα and expression of mda-9/syntenin were interdependent. First, mda-9/syntenin inhibition suppressed basal or FN-induced phosphorylation of PKCα at Thr638/641, whereas PKCα inhibition suppressed basal or FN-induced expression of mda-9/syntenin. Second, inhibiting either mda-9/syntenin or PKCα suppressed FN-induced formation of integrin-β1/FAK/c-Src signaling complexes. Third, inhibiting either mda-9/syntenin or PKCα suppressed FN-induced phosphorylation of FAK Tyr397 and c-Src Tyr416 and the induction of downstream effector signals to p38 and mitogen-activated protein kinase, Cdc42, and NF-κB. In summary, our findings offer evidence that mda-9/syntenin acts as a molecular adaptor linking PKCα and FAK activation in a pathway of FN adhesion by human breast cancer and melanoma cells. Cancer Res; 70(4); 1645–55

PDZ domain–containing proteins are ubiquitous scaffolding proteins that are involved in the organization of multiprotein complexes at the membrane (1, 2). mda-9/syntenin is a 32-kDa protein that is made up of a 113-amino-acid NH2-terminal domain with no obvious structural motifs, followed by two adjacent tandem PDZ domains (PDZ1 and PDZ2) and a short 24-amino-acid COOH-terminal domain (3). The PDZ domains of mda-9/syntenin are essential for the assembly and organization of diverse cell signaling processes occurring at the plasma membrane (4, 5) and bind to multiple peptide motifs with low to medium affinity (6, 7). This plasticity allows mda-9/syntenin to participate in multiple biological functions, including receptor clustering, protein trafficking, and activation of the transcription factor Sox4 (4, 5).

Our lab and others have shown that mda-9/syntenin is overexpressed in several cancer cells and tissues and may regulate tumor cell invasion and metastasis (812). mda-9/syntenin displays preferential association with fibronectin (FN) signaling in human melanoma cells (810). mda-9/syntenin is colocalized with focal adhesion kinase (FAK) and facilitates FN-induced phosphorylation of FAK, with subsequent activation of p38 and c-jun NH2-terminal kinase mitogen-activated protein kinases (8, 10). Activation of FAK and p38 mitogen-activated protein kinase by mda-9/syntenin leads to activation of nuclear factor κB (NF-κB; ref. 10). mda-9/syntenin may directly interact with c-Src, and this interaction correlates with increased formation of active FAK/c-Src signaling complexes (9) important for regulating the migration machinery (13).

Integrins are α and β heterodimeric cell-surface receptors that mediate cell extracellular matrix proteins (14). Ligand binding to the extracellular integrin domain induces conformational changes and integrin clustering for activation of signaling cascades and recruitment of multiprotein complexes to focal adhesions (15). Subsequently, integrins lacking kinase activity transmit messages through a variety of intracellular protein kinases and adaptor molecules such as FAK and paxillin (16, 17). FAK is a nonreceptor tyrosine kinase that regulates signal transduction at sites of integrin-mediated cell adhesions (18, 19). Clustering of integrins facilitates the autophosphorylation of FAK at Tyr397 through undefined mechanisms (20). Integrin-stimulated autophosphorylation creates a high-affinity binding site for the Src-homology 2 domain of Src family tyrosine kinases, and this interaction promotes Src kinase activity through a conformational change (21, 22). Activated c-Src binds to phosphorylated FAK at Tyr397 and then phosphorylates additional sites on FAK, which promotes the assembly of distinct and higher-order individual signaling complexes, thereby providing a mechanism for coordinating signaling through multiple pathways (19). In many signaling contexts, the FAK-Src complex controls cell shape and focal contact turnover events during cell motility (18, 19, 23). Protein kinase C (PKC) also regulates integrin-mediated FAK phosphorylation and focal adhesion formation in many cell types (2428). In particular, PKCα seems to be a key intermediate between integrins and FAK signaling. PKCα activation is upstream FAK phosphorylation in response to FN. Inhibition of PKCα suppresses focal adhesion formation, cell migration, and FAK phosphorylation at Tyr397 during adhesion to FN (26, 2931). However, the mechanism by which links between PKCα activation and FAK phosphorylation remains poorly understood.

Here, we report that the expression of mda-9/syntenin and activation of PKCα are interdependently regulated. Moreover, upregulation of mda-9/syntenin by PKCα facilitated the formation of the FN-induced assembly of signaling complexes including integrin β1, FAK, and c-Src, which activate the FAK and FAK signaling pathways in MDA-MB-231 and C8161 cells. Our findings suggest that mda-9/syntenin may link PKCα and FAK activation and assemble multimeric integrin-β1 signaling complexes to activate FAK in response to FN.

Cell culture

The human breast cancer cell lines MCF-7, T47D, and MDA-MB-231 were purchased from American Type Culture Collection and maintained in RPMI 1640. The human melanoma line C8161 was a gift from Dr. Danny R. Welch (The University of Alabama at Birmingham, Birmingham, AL) and was maintained in DMEM. MCF-7/Syn and MCF-7/Vec cells, which were stably transfected with a Flag-tagged mda-9/syntenin and the control vector, respectively, were previously described (11). These media were supplemented with penicillin-streptomycin (Invitrogen) and 10% heat-inactivated fetal bovine serum (Hyclone). All cells were maintained in a humidified 5% CO2 atmosphere at 37°C.

Antibodies and reagents

Antibody for mda-9/syntenin was purchased from Abnova. Antibodies for PKCα and PKCδ were from BD Biosciences. Antibodies for phospho-PKCα, phospho-PKCδ, FAK, phospho-p38, phospho-c-Src, c-Src, and phospho-extracellular signal–regulated kinase (Erk) were from Cell Signaling Technology. Antibody for phospho-FAK was from Stressgen, and antibodies for α-tubulin and Flag were from Sigma. Protein A/G plus-agarose beads and antibodies for integrin β1, hemagglutinin (HA), ErbB2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Santa Cruz Biotechnology. Actinomycin D, PP2, Gö6976, and rottlerin were from Calbiochem. Fibronectin, poly-l-lysine, and collagen type I were from BD Biosciences.

Plasmids and RNA interference

The expression vector for Flag-tagged mda-9/syntenin was described previously (11). The PKCα, PKCδ, dominant negative (DN)-PKCα, and DN-PKCδ constructs were kindly provided by Dr. J.J. Lee (KRIBB, Daejeon, Korea). The DN-Cdc42 construct was provided by Dr. K.Y. Lee (Chonnam National University, Kwangju, Korea). The expression vector for FAK-related nonkinase (FRNK) was a generous gift from D. Jeoung (Kangwon National University, Chuncheon, Korea). siRNAs for mda-9/syntenin, syndecan-2, syndecan-4, and scramble control were from Santa Cruz Biotechnology. Transfections were done using Lipofectamine Plus reagent according to the manufacturer's instructions (Invirogen).

Construction of mda-9/syntenin shRNA–producing vector

The pSUPER shRNA–producing plasmids (pSUPER-mda-9/syntenin and pSUPER-Scramble) were constructed according to the manufacturer's protocol (Oligoengine). The following target sequences were used: mda-9/syntenin, 5′-GCAAGACCTTCCAGTATAA-3′; scramble control: 5′-AATTCTCCGAACGTGTCACGT-3′. MDA-MB-231 cells were transfected with pSUPER-mda-9/syntenin or pSUPER-Scramble using the Lipofectamine Plus reagent. Forty-eight hours after transfection, the medium was changed with the culture medium supplemented with 500 μg/mL G418 for selection of stable transfectants.

Reverse transcription-PCR assays

To measure the RNA levels for mda-9/syntenin and PKCα, we used the following primers: mda-9/syntenin, 5′-TGAAAACTGTGCAGGATGGA-3′ (sense) and 5′-ACCTCAGGAATGGTGTGGTC-3′ (antisense); PKCα, 5′-TGTGCACCCCATCTTACAGGGTGCAGTATGA-3′ (sense) and 5′-TCATACTGCACCCTGTAAGATGGGGTGCACA-3′ (antisense). GAPDH was also amplified as the reference gene using the following primers: 5′-ACCACAGTCCATGCCATCAC-3′ (sense) and 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense).

Immunoprecipitation and Western blotting

Immnuoprecipitation and Western blotting were described previously (11, 32). Cells were lysed in lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L sodium orthovanadate, 1% NP40, and protease inhibitors cocktail (BD Biosciences)] and centrifuged at 15,000 rpm for 30 min at 4°C. For immunoprecipitation, equivalent amounts of cell lysates were incubated for 16 h at 4°C with the appropriate antibodies, followed by incubation with protein A/G-agarose beads for 2 h at 4°C. Immunoprecipitates were extensively washed, and the eluted precipitates were resolved by SDS-PAGE, transferred, and probed with the proper antibodies.

Cell fractionation

Cells were washed with PBS, incubated in hypotonic lysis buffer [50 mmol/L Tris-HCl (pH 7.0), 1 mmol/L EDTA, 0.1% β-mercaptoethanol, 5 mmol/L sodium orthovanadate, protease inhibitors cocktail], and lysed by 15 strokes of a prechilled 1-mL Dounce homogenizer with a tight-fitting pestle. Unbroken cells and nuclei were pelleted at 1,000 × g at 4°C for 10 min. The cytoplasmic fraction was obtained by centrifuging the supernatants at 21,000 × g at 4°C for 45 min, and the pellets containing cellular membranes were washed three times in hypotonic lysis buffer and resuspended in lysis buffer.

Cell migration and invasion assays

Cell migration and invasion assays were done as described previously (11, 32). Briefly, cells were seeded in triplicate at a density of 5 × 104 cells on the upper part of each chamber. Chambers were incubated at 37°C for 4 h (for migration) or 12 h (for invasion). The cells that had migrated to the lower surface of the filter were fixed with methanol. Then the cells were stained and counted in at least five randomly selected microscopic fields (×100) per filter.

NF-κB reporter assays

Briefly, MDA-MB-231 cells were transfected with a NF-κB–responsive luciferase reporter plasmid (Stratagene) in combination with the indicated constructs. Forty-eight hours after transfection, cells were trypsinized and plated on poly-l-lysine– or FN-coated surface in serum-free medium for 2 h. Luciferase assay was done using the dual-luciferase reporter assay system according to the manufacturer's instructions (Promega). The results were normalized to the activity of renilla expressed by cotransfected Rluc gene under the control of a constitutive promoter.

Immunofluorescence and confocal microscopy

Cells were rinsed once in PBS, fixed in fresh 4% paraformaldehyde for 15 min at room temperature, and permeabilized in 0.5% Triton X-100. Nonspecific sites were blocked by incubation with PBS containing 1% goat serum before incubating the cells with the appropriate antibody. After four washes in PBS, the cells were incubated with goat anti-mouse secondary Alexa 488 (for mda-9/syntenin) or goat anti-rabbit secondary Alexa 546 (for PKCα) for 1 h at room temperature and mounted. F-actin was stained with rhodamine-labeled phalloidin (Molecular probes) followed by four washes in PBS. Confocal images were acquired by using a Zeiss LSM510 META NLO inverted laser scanning confocal microscope (Carl Zeiss; Korea Basic Science Institute Chuncheon Center) equipped with an external argon, HeNe laser, and HeNe laser II. Using a C-Apochromat 63× (numerical aperture 1.2) water immersion objective (Zeiss), images were captured at the colony midsection.

CDC42 activity ELISA

A commercially available Cdc42 ELISA was used according to the manufacturer's instructions (Cytoskeleton, Inc.). Briefly, captured Cdc42 was cross-linked to the plate and detected by sequential incubations with an anti-Cdc42 primary antibody and a horseradish peroxidase–conjugated secondary antibody. The relative amount of bound Cdc42 was then measured by developing the plate with a horseradish peroxidase–sensitive dye, stopping the development with dilute sulfuric acid, and recording the absorbance at 490 nm using a microplate reader.

Statistical analysis

The data are expressed as the mean ± SD unless otherwise specified. Statistical significance was assessed by two-tailed unpaired Student's t test and P < 0.05 was considered statistically significant.

mda-9/syntenin expression is dependent on the activation of PKCα during adhesion to FN in MDA-MB-231 and C8161 cells

To investigate the molecular mechanism of mda-9/syntenin for inducing cell migration and invasion, we analyzed the effect of FN stimulation on mda-9/syntenin expression and PKCα, c-Src, and FAK activation in MDA-MB-231 and C8161 cells (Fig. 1A), which have high endogenous mda-9/syntenin expression levels (8, 11). mda-9/syntenin expression was significantly induced by FN stimulation. The expression level of mda-9/syntenin was increased within 30 min and reached a peak at 1 hour, and then this induction returned to basal level 8 hours after FN stimulation. The levels of phospho-PKCα and total PKCα were also increased after FN stimulation in a manner similar to mda-9/syntenin induction. FAK Tyr397 phosphorylation and c-Src Tyr416 phosphorylation were significantly induced 1 hour after FN stimulation, suggesting that both mda-9/syntenin induction and PKCα activation could precede FAK and c-Src activation. PKCδ, FAK, and c-Src expression and PKCδ phosphorylation levels were not modulated by FN stimulation. mda-9/syntenin levels and PKCα phosphorylation were not changed in response to collagen type I (Fig. 1B). Furthermore, FAK phosphorylation was not significantly increased by collagen type I stimulation.

Figure 1.

PKCα-dependent induction of syntenin during adhesion to FN. A, MDA-MB-231 and C8161 cells were serum starved for 12 h and allowed to adhere to a FN-coated plate (10 μg/mL) for the indicated periods of time in absence of serum. Whole-cell lysates were blotted with the indicated antibodies. B, serum-starved MDA-MB-231 cells were allowed to adhere to a collgen type I–coated plate (10 μg/mL) for the indicated periods of time in absence of serum. Whole-cell lysates were blotted with the indicated antibodies. C, top, serum-starved MDA-MB-231 cells were plated onto FN for 1 h with Gö6976 (1 μmol/L), rottlerin (10 μmol/L), or PP2 (10 μmol/L) for 1 h. Bottom, serum-starved MDA-MB-231 cells transfected with DN of PKCα or the control vector were plated onto FN for 1 h. Whole-cell lysates were used to determine the levels of syntenin by Western blotting. D, effect of PKCα on mda-9/syntenin–induced cell migration (left) and invasion (right). Left, MCF-7/Syn cells were transfected with DN-PKCα, DN-PKCδ, DN-Cdc42, or FRNK vector. Right, MDA-MB-231 cells were transfected with mda-9/syntenin–targeted siRNA, DN-PKCα, DN-PKCδ, DN-Cdc42, or FRNK vector. Assays were done as described in Materials and Methods. Columns, average of three independent experiments done in triplicate; bars, SD. *, P < 0.01.

Figure 1.

PKCα-dependent induction of syntenin during adhesion to FN. A, MDA-MB-231 and C8161 cells were serum starved for 12 h and allowed to adhere to a FN-coated plate (10 μg/mL) for the indicated periods of time in absence of serum. Whole-cell lysates were blotted with the indicated antibodies. B, serum-starved MDA-MB-231 cells were allowed to adhere to a collgen type I–coated plate (10 μg/mL) for the indicated periods of time in absence of serum. Whole-cell lysates were blotted with the indicated antibodies. C, top, serum-starved MDA-MB-231 cells were plated onto FN for 1 h with Gö6976 (1 μmol/L), rottlerin (10 μmol/L), or PP2 (10 μmol/L) for 1 h. Bottom, serum-starved MDA-MB-231 cells transfected with DN of PKCα or the control vector were plated onto FN for 1 h. Whole-cell lysates were used to determine the levels of syntenin by Western blotting. D, effect of PKCα on mda-9/syntenin–induced cell migration (left) and invasion (right). Left, MCF-7/Syn cells were transfected with DN-PKCα, DN-PKCδ, DN-Cdc42, or FRNK vector. Right, MDA-MB-231 cells were transfected with mda-9/syntenin–targeted siRNA, DN-PKCα, DN-PKCδ, DN-Cdc42, or FRNK vector. Assays were done as described in Materials and Methods. Columns, average of three independent experiments done in triplicate; bars, SD. *, P < 0.01.

Close modal

mda-9/syntenin mRNA expression was not altered after FN stimulation, as assessed by reverse transcription-PCR analysis (Supplementary Fig. S1A). Furthermore, pretreatment with actinomycin D did not change mda-9/syntenin and PKCα protein expression levels (Supplementary Fig. S1B). Rottlerin, a selective PKCδ inhibitor, or PP2, a selective Src kinase inhibitor, also did not significantly change FN-induced expression of mda-9/syntenin (Fig. 1C, top). In contrast, Gö6976, a selective inhibitor of PKCα, suppressed FN-induced mda-9/syntenin expression (Fig. 1C, top). Similarly, transfection of DN of PKCα blocked FN-induced mda-9/syntenin expression in MDA-MB-231 cells (Fig. 1C, bottom), suggesting that FN-induced mda-9/syntenin expression can be regulated at the posttranscriptional level in a PKCα-dependent manner.

mda-9/syntenin overexpression into MCF-7 cells induced cell migration toward FN, but this effect was blocked by the DN of PKCα, Cdc42, and FAK, but not by PKCδ (Fig. 1D, left). Similarly, invasiveness of MDA-MB-231 cells was also significantly suppressed by the DN of PKCα, Cdc42, FAK, and mda-9/syntenin siRNA (Fig. 1D, right). These results suggest that PKCα may regulate mda-9/syntenin–induced signaling in response to FN in these cells.

PKCα mediates mda-9/syntenin expression

Forced expression of DN of PKCα into MDA-MB-231 or C8161 cells resulted in a significant suppression of the endogenous expression of mda-9/syntenin (Fig. 2A). Moreover, PKCα phosphorylation in MDA-MB-231 cells was higher than that in T47D and MCF-7 cells (Fig. 2B), which have low endogenous mda-9/syntenin expression (11), suggesting that PKCα activity may regulate mda-9/syntenin expression.

Figure 2.

Interdependent regulation of mda-9/syntenin expression and PKCα activation. A, MDA-MB-231 and C8161 cells were transfected with the indicated amount of HA-tagged DN-PKCα. Whole-cell lysates were blotted with the indicated antibodies. B, Western blotting for mda-9/syntenin, PKCα, and phospho-PKCα expression in MDA-MB-231, MCF-7, and T47D cell lines. C, Western blotting for PKCα, phospho-PKCα, PKCδ, and phospho-PKCδ expression in MCF-7/Syn and MCF-7/Vec cells. D, MDA-MB-231 and C8161 cells were transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S). Whole-cell lysates were blotted with the indicated antibodies.

Figure 2.

Interdependent regulation of mda-9/syntenin expression and PKCα activation. A, MDA-MB-231 and C8161 cells were transfected with the indicated amount of HA-tagged DN-PKCα. Whole-cell lysates were blotted with the indicated antibodies. B, Western blotting for mda-9/syntenin, PKCα, and phospho-PKCα expression in MDA-MB-231, MCF-7, and T47D cell lines. C, Western blotting for PKCα, phospho-PKCα, PKCδ, and phospho-PKCδ expression in MCF-7/Syn and MCF-7/Vec cells. D, MDA-MB-231 and C8161 cells were transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S). Whole-cell lysates were blotted with the indicated antibodies.

Close modal

mda-9/syntenin overexpression in MCF-7 cells increased endogenous PKCα phosphorylation without affecting PKCδ (Fig. 2C). In contrast, transfection of mda-9/syntenin siRNA into MDA-MB-231 or C8161 cells reduced the endogenous levels of PKCα phosphorylation (Fig. 2D). Taken together, these results suggest that PKCα and mda-9/syntenin could be interdependently regulated in these cell lines.

mda-9/syntenin mediates FN-induced activation of PKCα

Transfection of C8161 cells with mda-9/syntenin siRNA, but not control siRNA, blocked FN-induced increases in PKCα phosphorylation, but did not change PKCδ phosphorylation (Fig. 3A). Similarly for MDA-MB-231 cells, mda-9/syntenin siRNA also blocked FN-induced phosphorylation of PKCα (Fig. 3B). Membrane targeting of PKCα is critical for PKCα activation (33). mda-9/syntenin shRNA blocked FN-induced membrane targeting of PKCα, but control shRNA did not, as assessed by immunofluorescence staining (Fig. 3C, left). We further confirmed this result with membrane fractionation (Fig. 3C, right), where knockdown of mda-9/syntenin again blocked the membrane targeting of PKCα after FN stimulation. Notably, most of mda-9/syntenin was detected in the membrane fraction on FN stimulation. Coimmunoprecipitation assays revealed that mda-9/syntenin physically associated with PKCα, but not with PKCδ, in HEK293T/17 cells (Supplementary Fig. S2), and FN increased this association in MDA-MB-231 cells (Fig. 3D, left). Double immunofluorescence revealed that colocalization of mda-9/syntenin with PKCα was significantly increased by FN stimulation (Fig. 3D, right). These results indicate that mda-9/syntenin could regulate FN-induced activation of PKCα.

Figure 3.

mda-9/syntenin mediates FN-induced activation of PKCα. A and B, serum-starved C8161 cells (A) and MDA-MB-231 cells (B) transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S) were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies. C, MDA-MB-231 cells stably transfected with control shRNA (C) or mda-9/syntenin–targeted shRNA (S) were plated onto FN for 1 h and immunostained with PKCα antibody (left) or fractionated into cytosol and plasma membrane fractions (right). Both fractions were blotted with the indicated antibodies. ErbB2 was used as the membrane protein marker and GAPDH as the cytosol protein marker. D, effect of FN stimulation on the association of mda-9/syntenin with PKCα. MDA-MB-231 cells transfected with HA-tagged PKCα and Flag-tagged mda-9/syntenin were plated onto FN for 1 h. Left, whole-cell lysates were immunoprecipitated, followed by blotting with the indicated antibodies. Right, fluorescent confocal micrographs of MDA-MB-231 cells plated onto FN for 1 h showing immunolocalization of PKCα and mda-9/syntenin.

Figure 3.

mda-9/syntenin mediates FN-induced activation of PKCα. A and B, serum-starved C8161 cells (A) and MDA-MB-231 cells (B) transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S) were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies. C, MDA-MB-231 cells stably transfected with control shRNA (C) or mda-9/syntenin–targeted shRNA (S) were plated onto FN for 1 h and immunostained with PKCα antibody (left) or fractionated into cytosol and plasma membrane fractions (right). Both fractions were blotted with the indicated antibodies. ErbB2 was used as the membrane protein marker and GAPDH as the cytosol protein marker. D, effect of FN stimulation on the association of mda-9/syntenin with PKCα. MDA-MB-231 cells transfected with HA-tagged PKCα and Flag-tagged mda-9/syntenin were plated onto FN for 1 h. Left, whole-cell lysates were immunoprecipitated, followed by blotting with the indicated antibodies. Right, fluorescent confocal micrographs of MDA-MB-231 cells plated onto FN for 1 h showing immunolocalization of PKCα and mda-9/syntenin.

Close modal

Either mda-9/syntenin or PKCα regulates FN-induced phosphorylation of FAK at Tyr397

Treatment with mda-9/syntenin siRNA, but not control siRNA, blocked FN-induced FAK Tyr397 phosphorylation in MDA-MB-231 cells induced by plating on FN (Fig. 4A). In contrast, MCF-7 cells stably transfected with mda-9/syntenin increased FN-induced FAK Tyr397 phosphorylation (Fig. 4B). Next, we tested whether PKCα influenced FN-induced FAK Tyr397 phosphorylation. Indeed, pretreatment with Gö6976 significantly suppressed FAK phosphorylation induced by FN, but rottlerin did not (Fig. 4C). Similarly, forced expression of DN of PKCα suppressed FN-induced FAK phosphorylation, but the DN of PKCδ did not (Fig. 4D). Our results suggest that mda-9/syntenin may mediate FN-induced FAK Tyr397 phosphorylation in a PKCα-dependent manner, and that mda-9/syntenin and PKCα could function as upstream regulators of FAK activation.

Figure 4.

Inhibition of mda-9/syntenin or PKCα suppresses FN-induced activation of FAK. A, serum-starved MDA-MB-231 cells transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S) were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies. B, serum-starved MCF-7/Syn and MCF-7/Vec cells were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies. C, serum-starved MDA-MB-231 cells were plated onto FN for 1 h with Gö6976 (1 μmol/L; top) or rottlerin (10 μmol/L; bottom) for the indicated periods of time. Whole-cell lysates were blotted with the indicated antibodies. D, serum-starved MDA-MB-231 cells transfected with HA-tagged DN-PKCα (top) or DN-PKCδ (bottom) were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies.

Figure 4.

Inhibition of mda-9/syntenin or PKCα suppresses FN-induced activation of FAK. A, serum-starved MDA-MB-231 cells transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S) were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies. B, serum-starved MCF-7/Syn and MCF-7/Vec cells were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies. C, serum-starved MDA-MB-231 cells were plated onto FN for 1 h with Gö6976 (1 μmol/L; top) or rottlerin (10 μmol/L; bottom) for the indicated periods of time. Whole-cell lysates were blotted with the indicated antibodies. D, serum-starved MDA-MB-231 cells transfected with HA-tagged DN-PKCα (top) or DN-PKCδ (bottom) were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies.

Close modal

Either mda-9/syntenin or PKCα regulates FN-induced integrin β1/FAK/c-Src associations

Integrin β1 immunoprecipitated from unstimulated MDA-MB-231 cells weakly associated with FAK, c-Src, and mda-9/syntenin (Fig. 5A). However, FN stimulation increased the integrin-β1 association with FAK, c-Src, and mda-9/syntenin, and mda-9/syntenin siRNA blocked this effect (Fig. 5A). FN stimulation also increased the FAK association with integrin β1, c-Src, and mda-9/syntenin, but mda-9/syntenin siRNA blocked this effect (Fig. 5B). Similarly, forced expression of DN of PKCα impaired the formation of FN-induced integrin-β1 association with FAK, c-Src, and mda-9/syntenin (Fig. 5C), as well as FAK association with integrin β1, c-Src, and mda-9/syntenin (Fig. 5D). These results suggest that mda-9/syntenin could regulate FN-induced formation of signaling complexes, including integrin β1, FAK, and c-Src, through PKCα-mediated upregulation.

Figure 5.

Inhibition of mda-9/syntenin or PKCα suppresses FN-induced integrin β1/FAK/c-Src associations. A and B, serum-starved MDA-MB-231 transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S) were plated onto FN for 1 h. Whole-cell lysates were immunoprecipitated with integrin-β1 (A) or FAK antibody (B). The immunoprecipitates (IP) and whole-cell lysates (Input) were blotted with the indicated antibodies. C and D, serum-starved MDA-MB-231 cells transfected with HA-tagged DN-PKCα or control vector were allowed to adhere to a FN-coated plate for 1 h. Whole-cell lysates were immunoprecipitated with integrin-β1 (C) or FAK antibody (D). The immunoprecipitates (IP) and whole cell lysates (Input) were blotted with the indicated antibodies.

Figure 5.

Inhibition of mda-9/syntenin or PKCα suppresses FN-induced integrin β1/FAK/c-Src associations. A and B, serum-starved MDA-MB-231 transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S) were plated onto FN for 1 h. Whole-cell lysates were immunoprecipitated with integrin-β1 (A) or FAK antibody (B). The immunoprecipitates (IP) and whole-cell lysates (Input) were blotted with the indicated antibodies. C and D, serum-starved MDA-MB-231 cells transfected with HA-tagged DN-PKCα or control vector were allowed to adhere to a FN-coated plate for 1 h. Whole-cell lysates were immunoprecipitated with integrin-β1 (C) or FAK antibody (D). The immunoprecipitates (IP) and whole cell lysates (Input) were blotted with the indicated antibodies.

Close modal

Inhibition of mda-9/syntenin or PKCα impairs the FN-induced FAK signaling pathways and the reorganization of actin cytoskeleton

We further investigated the role of mda-9/syntenin and PKCα in FAK-mediated signaling pathways toward FN (19). FN stimulation significantly increased the levels of phosphorylation of Erk1/2, p38, and c-Src in MDA-MB-231 and C8161 cells (Supplementary Fig. S3). FN-induced phosphorylation levels of these kinases were blocked by DN of PKCα (Fig. 6A). mda-9/syntenin siRNA also blocked this phosphorylation (Fig. 6B), as did overexpression of FRNK (Supplementary Fig. S4). FN induced Cdc42 activation, but mda-9/syntenin siRNA or DN of PKCα blocked this activity (Fig. 6C, top). Consistently, mda-9/syntenin siRNA blocked FN-induced reorganization of actin cytoskeleton and formation of filopodia (Fig. 6C, bottom). Moreover, mda-9/syntenin siRNA or DN of PKCα blocked FN-induced NF-κB activation (Fig. 6D). These results further confirmed that mda-9/syntenin and PKCα could be critical regulators of FAK activation in response to FN.

Figure 6.

Inhibition of mda-9/syntenin or PKCα suppresses FN-induced activation of c-Src, p38, Erk, Cdc42 GTPase, and NF-κB. A, serum-starved MDA-MB-231 cells transfected with DN-PKCα were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies. B, whole-cell lysates were prepared from MDA-MB-231 cells transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S), as described in A, and blotted with the indicated antibodies. C, top, serum-starved MDA-MB-231 cells transfected with the indicated constructs were plated onto FN for 30 min, and Cdc42 activity was determined as described in Materials and Methods. Columns, average of three independent experiments done in triplicate; bars, SD. *, P < 0.01. Bottom, serum-starved MDA-MB-231 cells transfected with mda-9/syntenin–targeted siRNA or control siRNA were allowed to adhere to FN-coated coverslips (10 μg/mL) for 1 h and stained with rhodamine-labeled phalloidin for detection of F-actin filaments. D, MDA-MB-231 cells transfected with the indicated constructs were plated onto FN for 2 h, and NF-κB activity was measured as described in Materials and Methods. Columns, average of three independent experiments done in triplicate; bars, SD. *, P < 0.01.

Figure 6.

Inhibition of mda-9/syntenin or PKCα suppresses FN-induced activation of c-Src, p38, Erk, Cdc42 GTPase, and NF-κB. A, serum-starved MDA-MB-231 cells transfected with DN-PKCα were plated onto FN for 1 h. Whole-cell lysates were blotted with the indicated antibodies. B, whole-cell lysates were prepared from MDA-MB-231 cells transfected with control siRNA (C) or mda-9/syntenin–targeted siRNA (S), as described in A, and blotted with the indicated antibodies. C, top, serum-starved MDA-MB-231 cells transfected with the indicated constructs were plated onto FN for 30 min, and Cdc42 activity was determined as described in Materials and Methods. Columns, average of three independent experiments done in triplicate; bars, SD. *, P < 0.01. Bottom, serum-starved MDA-MB-231 cells transfected with mda-9/syntenin–targeted siRNA or control siRNA were allowed to adhere to FN-coated coverslips (10 μg/mL) for 1 h and stained with rhodamine-labeled phalloidin for detection of F-actin filaments. D, MDA-MB-231 cells transfected with the indicated constructs were plated onto FN for 2 h, and NF-κB activity was measured as described in Materials and Methods. Columns, average of three independent experiments done in triplicate; bars, SD. *, P < 0.01.

Close modal

The major conclusions of this study are as follows: (a) mda-9/syntenin and PKCα activation are significantly induced before FAK Tyr397 phosphorylation during adhesion to FN; (b) upregulation of mda-9/syntenin and activation of PKCα are interdependent, that is, inhibition of PKCα suppressed both FN-induced and endogenous expression of mda-9/syntenin, and vice versa; (c) inhibition of mda-9/syntenin or PKCα completely suppressed both the association of integrin β1/FAK/c-Src signaling complexes and the phosphorylation of FAK at Tyr397 and c-Src at Tyr416; and (d) mda-9/syntenin and PKCα inhibition suppressed FN-induced cell migration and invasion, as well as FAK-mediated signaling pathways. Collectively, our results provide the first evidence that mda-9/syntenin and PKCα are interdependently regulated, and PKCα-dependent upregulation of mda-9/syntenin could facilitate the associations of integrin β1/FAK/c-Src signaling complexes during adhesion to FN, leading to FAK and c-Src activation.

We showed that upregulation of mda-9/syntenin and activation of PKCα are interdependent, that is, inhibition of PKCα suppresses FN-induced and endogenous expression of mda-9/syntenin and inhibition of mda-9/syntenin impairs FN-induced activation of PKCα. Modulation of mda-9/syntenin expression by siRNA or overexpression significantly affected the activation of PKCα, FAK, and c-Src. Inhibition of PKCα also significantly suppressed FN-induced activation of FAK and c-Src, as well as mda-9/syntenin expression. Moreover, mda-9/syntenin and PKCα regulated the FN-induced association of integrin β1/FAK/c-Src signaling complexes. Therefore, FN-induced activation of PKCα induces the expression of mda-9/syntenin, which in turn could induce FAK activation by facilitating the formation of integrin β1/FAK/c-Src signaling complexes. mda-9/syntenin may act as a molecular adaptor that links PKCα and FAK during adhesion to FN.

PKCα is essential for integrin-mediated adhesion, migration, and signaling events through participation in integrin β1–associated protein complexes downstream of FN (2831, 34). In particular, binding of integrin α5β1 to FN activates PKCα, and inhibition of PKCα, but not PKCδ, suppresses focal adhesion formation and cell migration mediated by α5β1 (31). FN binding to integrin α5β1 requires a proteoglycan coreceptor, syndecan-4, for focal adhesion formation and cell migration (2931). Syndecan-4–dependent activation of PKCα is essential for focal adhesion formation and cell migration in cells that adhere to FN through integrin α5β1 (31). Consistent with these previous reports, our results showed that inhibition of PKCα significantly suppressed FN-induced cell migration and activation of FAK in MDA-MB-231 and C8161 cells. Moreover, downregulation of syndecan-4 by siRNA impaired FN-induced activation of PKCα and FAK as well as mda-9/syntenin expression in MDA-MB-231 cells (Supplementary Fig. S5), confirming that syndecan-4–dependent PKCα activation is essential for the activation of FAK during adhesion to FN in these cell lines. Therefore, it is possible to speculate that syndecan-4–dependent activation of PKCα induces mda-9/syntenin expression, which in turn facilitates FN-induced activation of FAK.

These findings raise intriguing questions about how mda-9/syntenin activates PKCα, which in turn regulates the expression of mda-9/syntenin and facilitates the associations of integrin β1/FAK/c-Src signaling complexes. Syndecan-4 cytoplasmic domain binds both phosphatidylinositol 4,5-bisphosphate (PIP2) and PKCα and may potentiate the activation of PKCα (29, 35). mda-9/syntenin was originally identified as a binding protein for the cytoplasmic domain of syndecan including syndecan-4 (36). PDZ domains of mda-9/syntenin can bind to PIP2 (37). Point mutations of amino acids required for PIP2-binding completely abolish mda-9/syntenin–induced cell invasion, suggesting that the binding of mda-9/syntenin and PIP2 could play a critical role in mda-9/syntenin–induced signaling activation (12). PKCα can bind to PIP2 through its C2 domain for plasma membrane localization (38, 39). Our results showed that mda-9/syntenin could physically associate with PKCα, and FN stimulation significantly increased the association between mda-9/syntenin and PKCα. Moreover, inhibition of mda-9/syntenin impaired the plasma membrane targeting of PKCα. Therefore, mda-9/syntenin may facilitate the binding of PKCα to PIP2 through the formation of mda-9/syntenin/syndecan-4/PKCα complexes at the plasma membrane after FN attachment. Inhibition of PKCα suppressed both endogenous and FN-induced expression of mda-9/syntenin. Expression of mda-9/syntenin is probably regulated by a positive feedback mechanism from PKCα activation. Further studies are needed to fully understand how mda-9/syntenin activates PKCα, which in turn regulates the expression of mda-9/syntenin.

We here showed that mda-9/syntenin regulates the associations of integrin β1, FAK, and c-Src, and it may function as an adaptor for the assembly of integrin-β1 signal complexes including FAK and c-Src during adhesion to FN. Consequently, the increased expression of mda-9/syntenin would result in the increase of its ability to assemble with sufficient efficiency to integrin β1–associated signaling complexes, leading to activation of FAK and c-Src, and would therefore have a profound effect on integrin β1-FAK signaling pathways. The detailed mechanisms by which mda-9/syntenin assembles the FN-induced integrin-β1 signaling complexes remain to be elucidated.

In summary, activation of PKCα induces expression of mda-9/syntenin, which in turn enhances the FN-induced assembly of integrin-β1 signaling complexes with FAK and c-Src, activating FAK and its downstream pathways. This may explain the relationship between activation of PKCα and formation of focal adhesions to FN (2831). Our findings also support the hypothesis that mda-9/syntenin functions as an adaptor protein for the FN-induced assembly of multimeric signaling complexes clustered at high concentrations on the plasma membrane. The activated mda-9/syntenin/PKCα/integrin-β1/FAK/c-Src complexes may then enhance cell migration, invasion, and metastasis. Targeted therapies that inactivate the mda-9/syntenin/PKCα/integrin-β1/FAK/c-Src complexes may provide a rational molecular approach to improve the prognosis of cancer patients.

No potential conflicts of interest were disclosed.

Grant Support: The Korea Healthcare Technology R&D Project (A084250) and the Ministry for Health, Welfare and Family Affairs.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data