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Cancer Research 67, 10309, November 1, 2007. doi: 10.1158/0008-5472.CAN-07-3256
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Hepatocyte Growth Factor–Mediated Cell Invasion in Pancreatic Cancer Cells Is Dependent on Neuropilin-1

Arikira Matsushita, Tobias Götze and Murray Korc

Departments of Medicine, Pharmacology, and Toxicology, and the Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center and Dartmouth Medical School, Hanover, New Hampshire

Requests for reprints: Murray Korc, Department of Medicine, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756. Phone: 603-650-7936; Fax: 603-650-6122; E-mail: murray.korc{at}dartmouth.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropilin-1 (Np-1), a receptor for semaphorin 3A and vascular endothelial growth factor, is expressed at high levels in pancreatic ductal adenocarcinoma (PDAC). To assess the potential role of Np-1 in PDAC, COLO-357 pancreatic cancer cells, which express relatively low levels of Np-1, were stably transfected with the Np-1 cDNA. Np-1 overexpression was associated with enhanced cell invasiveness in response to hepatocyte growth factor (HGF), and this effect was abolished by small interfering RNA–mediated down-regulation of c-Met. Conversely, in PANC-1 pancreatic cancer cells, which express relatively high levels of Np-1, suppression of endogenous Np-1 completely abolished HGF-mediated cell invasion. To determine which pathways are involved in Np-1–mediated facilitation of c-Met–dependent cell invasiveness, the effects of HGF on signaling were examined next in sham-transfected and Np-1–overexpressing COLO-357 cells. HGF actions on c-Met tyrosine phosphorylation and p38 mitogen-activated protein kinase (MAPK) activation were increased in Np-1–overexpressing COLO-357 cells by comparison with HGF effects in sham-transfected cells. SB203580, an inhibitor of p38 MAPK, suppressed HGF-induced invasion in Np-1–overexpressing cells, whereas U0126, a MAP/extracellular signal-regulated kinase kinase inhibitor, was without effect. PP2, a Src inhibitor, and LY294002, a phosphatidylinositol 3-kinase inhibitor, also suppressed HGF-induced invasion in these cells. Immunoprecipitation studies revealed that Np-1 associated with c-Met, but not with epidermal growth factor receptor, family members. Confocal microscopy indicated that this association occurred on the plasma membrane and that HGF promoted the internalization of Np-1–c-Met complex, leading to its perinuclear localization. These findings indicate that Np-1 is required for efficient activation of c-Met–dependent pathways that promote cell invasiveness. [Cancer Res 2007;67(21):10309–16]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that is characterized by a high rate of mutations in the K-ras oncogene, the p53 and Smad4 tumor suppressor genes, and the p16 cell cycle–regulating gene (1, 2). In addition, the cancer cells in PDAC overexpress multiple transmembrane receptors and their ligands (3). Both non–heparin-binding growth factors, such as epidermal growth factor (EGF) and transforming growth factor-{alpha} (TGF-{alpha}), and heparin-binding growth factors, such as hepatocyte growth factor (HGF), vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-1 (FGF-1), FGF-2, and FGF-5, are overexpressed in PDAC (3–6).

Neuropilin-1 (Np-1), originally identified as a mediator of chemorepulsive guidance for axons in the developing nervous system (7, 8), is expressed at high levels in the cancer cells in PDAC (9, 10). Np-1 is a nontyrosine kinase transmembrane protein that acts as a coreceptor for VEGF-A (11). Its extracellular region consists of two complementing binding-like domains (a1 and a2), two coagulation factor V/VIII homology domains (b1 and b2), and a meprin A5 (MAM or c) domain, whereas its intracellular domain consists of a short cytoplasmic tail of about 40 amino acids (7, 8). Neuropilin-2 (Np-2) has a similar domain structure, with an overall sequence homology between the two genes of 44% (12).

Np-1 has been implicated in promoting cell survival and acts as a coreceptor with respect to the interactions of heparin-binding growth factors with their cognate high-affinity receptors (13). Np-1 also acts as a coreceptor for several class 3–secreted semaphorins (7, 8). Some of these secreted semaphorins also bind to Np-2, and some bind to both Np-1 and Np-2 (14). Semaphorins also bind to plexins, which are transmembrane receptors that are related to semaphorins (15). The human plexins were originally cloned as a result of their homology to the MET oncogene, which exhibits 10% homology to classic Sema domains (16). c-Met is a receptor tyrosine kinase that binds HGF and that has been implicated in cancer cell survival and invasiveness (17). It is not known, however, whether c-Met activation by HGF is modulated by interactions with Np-1.

Both c-Met and HGF are overexpressed in PDAC (18), and activation of the c-Met signaling pathways has been proposed to promote cancer cell invasiveness (19). The purpose of the present study was to determine whether Np-1 modulates HGF actions on cell invasiveness and, if so, to assess the mechanisms by which this modulation is effected. We now report that Np-1 enhances the ability of HGF to promote cell invasiveness by associating with c-Met that this association leads to increased HGF-mediated activation of p38 MAPK pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. The following items were purchased: fetal bovine serum (FBS) from Omega Scientific; DMEM, trypsin solution, and penicillin, streptomycin, and amphotericin B from Media Tech; LipofectAMINE PLUS reagent kit from Invitrogen; ExGen 500 from Fermentas; anti–Np-1 mouse monoclonal (A-12), anti–c-Met (C-12), and anti-HA (Y-11) rabbit polyclonal antibodies and protein-protein A/G plus agarose from Santa Cruz Biotechnology; anti–c-Met mouse monoclonal (clone 25H2) and anti–human EGF receptor (EGFR)-1 (HER1) rabbit polyclonal antibodies from Cell Signaling; anti–human erbB-3 (HER3; clone 2F12), anti–c-Met mouse monoclonal (DQ-13), and anti–human erbB-2 (HER2) rabbit polyclonal antibodies from Upstate Biotechnology; anti-HA mouse monoclonal antibody (clone 12CA5) from Roche Applied Science; anti–phosphorylated MAPK and anti–phosphorylated p38 MAPK rabbit polyclonal antibodies from Promega; antiphosphotyrosine mouse monoclonal antibody (PY20) from BD Transduction Laboratories; Alexa Fluor 555-conjugated or 488-conjugated goat anti-rabbit and anti-mouse secondary antibodies and Hoechst 33258 dye from Molecular Probes; Immobilon P membrane from Millipore; small interfering RNA (siRNA) nucleotides targeting c-Met mRNA and RISC-free scrambled siRNA from Dharmacon; glutathione S-transferase (GST)–PBD from Cytoskeleton, Inc.; immobilized anti-HA, Super Signal detection system and bicinchoninic acid protein assay kit from Pierce; Bio-Max film from Eastman Kodak; horseradish-peroxidase–conjugated anti-mouse and anti-rabbit antibodies from Bio-Rad Laboratories; PP2, SB203580, and U0126 from Alexis Biochemicals; LY294002 from Calbiochem; mitomycin C and all other reagents from Sigma Chemical Corp.; human recombinant HGF from R&D Systems; PANC-1 and COS-7 cells from the American Type Culture Collection. COLO-357 cells were a gift from R.S. Metzgar (Duke University, Durham, NC).

Cell culture. COLO-357, PANC-1, and COS-7 cells were grown in DMEM with 8% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin, 250 ng/mL Amphotericin B. Cells were maintained at 37°C in a humid atmosphere with 5% CO2.

Construction and transfection of expression plasmids. The Np-1 full-length cDNA was subcloned into pMH vector, which expresses a C-ternimal HA tag fusion protein. Authenticity was confirmed by sequencing. COS-7 cells were transiently transfected in serum-free medium with the vector using LipofectAMINE PLUS. COLO-357 cells were transfected with ExGen 500 and stably transfected clones were selected with 400 µg/mL geneticin (G418) in DMEM. Individual clones were isolated, and expression of the HA-tagged Np-1 protein was determined by Western blotting with anti–Np-1 (A-12) and anti-HA (Y-11) antibodies. Preparation of the Np-1 antisense cDNA, its subcloning into pLNCX2 vector, and preparation of sham-transfected and Np-1 antisense–expressing clones (Np-1AS) of PANC-1 cells were previously reported (20). All stable lines were maintained in DMEM with 400 µg/mL G418.

Immunoblotting and immunoprecipitation. Cells were either cultured to subconfluency in 8% FBS or serum-starved for 24 h before treatments. Cells were washed in PBS and lysed in buffer containing 20 mmol/L Tris-HCl (pH 7.5), 0.1 mol/L NaCl, 5 mmol/L NaF, 1% Triton X-100, 1 mmol/L Na3VO4, 10 mmol/L Na4P2O7, and protease inhibitors (1 µg/mL leupeptin, pepstatin, and aprotinin and 1 mmol/L phenylmethylsulfonyl fluoride). After determination of the amount of protein in the cell lysates, samples were then fractionated through a 7.5% polyacrylamide gel (PAGE), electrophoretically transferred to Immobilon P membranes, and blotted as described previously (21). After blocking for 1 h at 23°C, the membranes were incubated overnight at 4°C with the indicated primary antibodies and for 60 min with the corresponding horseradish-conjugated secondary antibodies. Bound antibodies were visualized using enhanced chemiluminesence. All experiments were done at least twice with similar results.

For immunoprecipitation, 1 mg of lysate was incubated overnight at 4°C with anti-HA agarose or anti–c-Met antibody (25H2) followed by 2-h incubation with protein A/G plus agarose. The beads were then washed thrice with 0.05% Tween 20 TBS, then boiled and subjected to PAGE for immunoblotting.

Invasion assay. The invasiveness of sham-transfected and Np-1–overexpressing COLO-357 cells was measured as reported previously, with some modification (22). Briefly, 7.5 x 104 cells were suspended in 500 µL of serum-free medium [0.1% bovine serum albumin (BSA)] in the absence or presence of 10 µmol/L SB203580, 1 µmol/L U0126, 10 µmol/L PP2, or 10 µmol/L LY294002 and placed onto this upper compartment of Matrigel-coated transwell chambers (8-µm pore size, BioCoat Matrigel Invasion Chambers, Becton Dickinson Labware). The lower compartment was filled with 750 µL of medium containing 5% FBS in the absence or presence of 1 nmol/L HGF and inhibitors. After 20 h, cells on the upper surface of the filter were carefully removed with a cotton swab, and the membranes were fixed in methanol and stained with crystal violet. The cells that had migrated through the membrane to the lower surface of the filter were counted using a microscope.

Immunofluorescence and confocal analysis. Cells were plated onto chamber slides, allowed to adhere overnight, and serum-starved for 24 h before the addition of 1 nmol/L HGF for the indicated times. Slides were fixed for 10 min in 2% paraformaldehyde, and free aldehydes were quenched with 50 mmol/L NH4Cl in PBS for 10 min. Cells were then permeabilized in 0.1% Triton X-100 in PBS-2% BSA for 15 min. Slides were then incubated at 23°C for 1 h with anti–c-Met (C-12) and anti-HA (12CA5) antibodies. After washing by PBS, slides were incubated for 30 min with Alexa Fluor 555- or 488-conjugated goat anti-rabbit or anti-mouse secondary antibodies, and the nuclei were counterstained with 0.2 µg/mL Hoechst 33258 dye for 1 min at 23°C, as previously reported (23). Slides were next washed thrice in PBS and once in water and then mounted in PermaFlour. Immunofluorescence was detected and recorded using a LSM-510 laser-scanning microscope (Carl Zeiss).

c-Met siRNA and transfection. siRNA nucleotides targeting c-Met mRNA and the corresponding RISC-free scrambled sequence were used according to the manufacturer's instructions. Briefly, a pool of four c-Met–specific 21 nucleotide RNA oligonucleotides were used in combination. siRNA duplexes were transiently transfected into the sham and Np-1–overexpressing COLO-357 cells using the ExGen 500 according to the manufacturer's instructions. RISC-free siRNA-transfected cells served as a negative control.

MAPK and p38 MAPK activation. Sham and Np-1–overexpressing cells were incubated in serum-free medium overnight, before the addition of 1 nmol/L HGF for the specified times, and washed in PBS. Cells were then lysed using the above lysis buffer. Lysates were resolved on 10% polyacrylamide gels, electrophoretically transferred to membranes, and immunoblotted with an anti–phosphorylated MAPK and anti–phosphorylated p38 MAPK antibodies.

Rac activation. Activated Rac was determined as described previously (24). To this end, cells were in serum-free medium for 24 h and stimulated with 1 nmol/L HGF for the times indicated. After stimulation, cells were washed with PBS and lysed using the lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl2, 0.3 mol/L NaCl, 2% IGEPAL]. Lysates were clarified by centrifuge for 5 min at 8,000 rpm at 4°C, and supernatants were removed. Two milligrams of lysate was incubated with the Rac-GTP–binding domain of PAK as a GST fusion protein (GST-PBD) for 1 h at 4°C. The beads were then washed with 0.05% Tween 20 TBS, boiled and subjected to 15% SDS-PAGE gels for immunoblotting with Thr anti-Rac antibody.

Statistics. Statistical significance was determined using ANOVA, followed by Tukey's HSD test of the comparisons that were deemed significant by ANOVA, with P < 0.05 taken as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of HGF on cell invasion. A hallmark of HGF action is its ability to promote cell invasion (25, 26). To determine whether Np-1 contributes to HGF-mediated cell invasiveness, COLO-357 cells, which express relatively low levels of endogenous Np-1, were stably transfected with the full-length Np-1 cDNA. Western blot analysis showed high levels of Np-1 protein in Np-1–transfected cells compared with sham cells, in which Np-1 was below the level of detection (Fig. 1A ). In sham-transfected COLO-357 cells, 1 nmol/L HGF did not significantly increase cell invasion, whereas in the Np-1–overexpressing cells, HGF caused a dramatic increase in cell invasion (Fig. 1B). However, elevated Np-1 levels per se did not lead to a significant increase in invasion in the absence of HGF (Fig. 1).


Figure 1
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  		Figure 1. Effects of HGF on cell invasion. A, Np-1 expression. Total cell lysates (30 µg per lane) from sham-transfected cells (Sham) and Np-1–overexpressing COLO-357 clones C1 and C2 were subjected to immunoblotting with an anti–Np-1 antibody (A-12). The blots were stripped and probed for ERK2 to assess protein loading. B, cell invasion. Cells were seeded on Matrigel-coated inserts in 24-well invasion chambers. The lower compartments were filled with the medium containing 5% FBS in the presence of either carrier buffer (Control) or 1 nmol/L HGF. After 20 h, the membranes were fixed in methanol and stained with crystal violet, and the cells that had migrated through the membranes were counted. Columns, means of duplicate determinations from three separate experiments; bars, SE. P = 0.016 (*) and P < 0.001 (**) when compared with respective controls; P = 0.014 ({dagger}) and P < 0.001 ({dagger}{dagger}) when compared with sham-transfected cells in the presence of HGF.

 
To determine whether HGF acted through its cognate receptor c-Met, the cells were next transfected with c-Met–siRNA, resulting within 72 h in a 60% to 90% decrease in c-Met protein levels in both sham-transfected and Np-1–overexpressing COLO-357 cells (Fig. 2A ). HGF (1 nmol/L) again failed to significantly increase cell invasion in sham-tranfected cells, but markedly increased invasion in Np-1–overexpressing cells that were transfected with the scrambled siRNA (Fig. 2B), to a level comparable with that observed in cells not transfected with the scrambled siRNA (Fig. 1B). By contrast, c-Met–siRNA–mediated down-regulation of c-Met protein in Np-1–overexpressing cells completely blocked HGF-induced invasion in clones C1 and C2 (Fig. 2B), confirming that the effects of HGF in the Np-1–overexpressing cells were mediated by c-Met.


Figure 2
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  		Figure 2. Effects of c-Met siRNA on cell invasion. A, c-Met immunoblotting. Cell lysates (30 µg per lane) were prepared from sham-transfected cells and Np-1–overexpressing COLO-357 clones C1 and C2 that were transfected with scrambled siRNA (–) or 200 nmol/L c-Met siRNA (+). Cells were subjected to immunoblotting with an anti–c-Met antibody. The membrane was probed for ERK2 to assess protein loading. B, effects of c-Met siRNA on HGF-induced cell invasion. Scrambled or c-Met siRNA-transfected cells that were either sham-transfected or Np-1–overexpressing COLO-357 clones C1 and C2 were seeded on the top chambers of Matrigel-coated 24-well plates. The bottom compartments were filled with medium containing 5% FBS supplemented with buffer (open columns) or 1 nmol/L HGF (filled columns). After 20 h, cells that had migrated through the membranes to the lower surface of the filter were counted. Columns, means of duplicate determinations from three separate experiments; bars, SE. P < 0.05 (*) and P < 0.001 (**) when compared with respective controls.

 
Effects of decreasing Np-1 levels in PANC-1 cells on invasion. PANC-1 human pancreatic cancer cells express relatively high levels of Np-1 (9). To determine whether endogenous Np-1 is required for HGF-mediated invasion in these cells, we next compared the effects of HGF on cell invasion in sham-transfected PANC-1 cells and in cells that were stably transfected with the Np-1 antisense cDNA (Np-1AS), which were previously shown to exhibit a marked decrease in endogenous Np-1 levels (20). In the absence of HGF, suppression of Np-1 did not significantly alter the invasiveness of PANC-1 cells (Fig. 3 ). However, the stimulatory effect of HGF on cell invasion in sham-transfected PANC-1 cells was completely abolished in the Np-1 antisense–expressing cells (Fig. 3). Thus, as was the case in COLO-357 cells, HGF-mediated invasion in PANC-1 cells required the presence of Np-1.


Figure 3
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  		Figure 3. Effects of HGF on invasion in PANC-1 cells. Sham-transfected (PANC-1 Sham) or Np-1 antisense expressing (PANC-1Np1-AS) cells were seeded on Matrigel-coated inserts in 24-well invasion chambers. The bottom compartments were filled with the medium containing 5% FBS, in the presence of either carrier buffer (control) or 1 nmol/L HGF, and the cells that had migrated through the membranes were counted as indicated in Fig. 1. Columns, means of duplicate determinations from three separate experiments; bars, SE. *, P < 0.02 when compared with control. Values for control sham-transfected cells were not significantly different from values for control or HGF-stimulated Np-1 antisense–expressing cells.

 
Effects of HGF on c-Met tyrosine phosphorylation and signaling. c-Met is a tyrosine kinase receptor that undergoes tyrosine autophosphorylation after HGF binding, leading to the activation of a complex signaling cascade, including activation of ras, extracellular signal-regulated kinase (ERK), src, and phosphatidylinositol 3-kinase (PI3K) signaling (27). To determine whether Np-1 levels altered HGF-mediated activation of c-Met and its downstream signaling pathways, the effects of HGF on c-Met tyrosine phosphorylation, ERK, and p-38 MAPK activation were examined next in sham-transfected and Np-1–overexpressing COLO-357 cells. HGF (1 nmol/L) addition caused maximal stimulation of c-Met tyrosine phosphorylation within 5 min, and this stimulation persisted for 30 min (data not shown). The 5-min time point was chosen, therefore, to compare the effects of HGF in sham-transfected and Np-1–overexpressing cells. HGF (1 nmol/L) caused a slight increase in c-Met tyrosine phosphorylation in sham-transfected cells, but a marked increase in tyrosine phosphorylation in Np-1–overexpressing cells (Fig. 4A ). The same concentration of HGF caused a rapid increase in phosphorylated ERK in sham-transfected cells, maximal stimulation occurring at 5 to 15 min after ligand addition (Fig. 4B). By contrast, the effects of HGF on phosphorylated ERK were markedly attenuated in the Np-1–overexpressing cells (Fig. 4B). Conversely, the appearance of phosphorylated p38 MAPK, which was maximal at 5 min after HGF addition, was markedly increased in the Np-1–overexpressing cells by comparison with the sham-transfected cells, and this increase persisted for the entire 60-min incubation period (Fig. 4B).


Figure 4
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  		Figure 4. Effects of HGF on phosphorylation of c-Met and downstream signaling. A, c-Met tyrosine phosphorylation. Sham-transfected cells and Np-1–overexpressing COLO-357 clones C1 and C2 were serum-starved for 24 h and treated with 1 nmol/L HGF for 5 min. Total cell lysates were immunoprecipitated with a c-Met antibody (25H2). Western blot analysis was then done using an anti-phosphotyrosine antibody. Equivalent loading of lanes was confirmed with Ponceau S staining. B, effects of HGF on activation of MAPK and p38 MAPK. The indicated cells were treated for the indicated times with 1 nmol/L HGF. Cell lysates (30 µg epr lane) were subjected to immunoblotting with antibodies against phosphorylated MAPK (pMAPK) and phosphorylated p38 MAPK (pP38). The blots were then stripped and probed for ERK2 to assess protein loading.

 
Effect of signaling inhibitors on HGF-induced invasion. To further assess the potential role of specific signaling pathways in cell invasion, the effects of SB203580, a selective inhibitor of p38 MAPK, U0126, a selective MEK inhibitor, PP2, a Src inhibitor, and LY294002, a PI3K inhibitor, on the invasion of sham and Np-1–overexpressing COLO-357 cells were examined next (Fig. 5 ). In sham-transfected cells, HGF did not significantly enhance cell invasion in the absence or presence of 10 µmol/L SB203580 (Fig. 5A). PP2 and LY294002 also failed to alter invasion in these cells (Fig. 5B), whereas U0126 (1 µmol/L), the highly selective MEK inhibitor, conferred onto HGF the ability to enhance invasion in these cells (Fig. 5B). Inasmuch as at this concentration U0126 completely suppresses the appearance of phosphorylated ERK in COLO-357 cells (28), these observations suggest that ERK activation by HGF may interfere with the ability of HGF to enhance invasion in COLO-357 cells.


Figure 5
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  		Figure 5. Effect of signaling inhibitors on HGF-induced cell invasion. A, effects of SB203580. Sham-transfected and Np-1–overexpressing COLO-357 cells (clone C2) were placed in 24-well invasion chambers in the absence or presence of 1 nmol/L HGF and/or 10 µmol/L SB203580. After 20 h, the cells that migrated through the membranes to the bottom surface of the filter were counted. B, effects of U0126, PP2 and LY294002. The above studies were repeated using 1 µmol/L U0126, 10 µmol/L PP2, and 10 µmol/L LY294002 in the absence or presence of 1 nmol/L HGF. Columns, means of duplicate determinations from three separate experiments; bars, SE. *, P < 0.01 and **, P < 0.001 when compared with respective controls.

 
By contrast to the findings in the sham-transfected cells, in Np-1–overexpressing cells, SB203580 (10 µmol/L) completely attenuated the marked HGF-induced increase in invasion (Fig. 5A), whereas U0126 did not alter the stimulatory effect of HGF on invasion in these cells (Fig. 5B). Thus, in the presence of Np-1, the ability of HGF to stimulate cell invasiveness is mediated via activation of p38 MAPK. Moreover, PP2 and LY294002 also completely abolished HGF actions on invasion in Np-1–overexpressing cells (Fig. 5B), indicating that src and PI3K-dependent pathways are also required for HGF-mediated stimulation of cell invasion.

Effects of HGF on Rac activation. Rac, a member of Rho family of small GTP-binding protein, has been implicated in stimulating cell motility by controlling actin polymerization into lamellipoidal membrane protrusions (29). To assess the potential role of Rac activation on HGF-mediated cell invasion, Rac activation assays were done next. Total Rac protein and activity levels were increased in Np-1–overexpressing cells when compared with sham-transfected cells. However, HGF did not alter Rac expression or activity either in sham-transfected or Np-1–overexpressing cells (Fig. 6 ).


Figure 6
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  		Figure 6. Effect of HGF on activation of Rac. Sham-transfected and Np-1–overexpressing COLO-357 cells (clone C2) were incubated for the indicated times with 1 nmol/L HGF. Activated Rac was pulled down using a GST fusion protein. Precipitates and total cell lysates (30 µg per lane) were subjected to immunoblotting with an anti-Rac antibody, and the membrane was reprobed for ERK2 to assess protein loading.

 
Np-1 interactions with c-Met. In view of the ability of Np-1 to modulate HGF-dependent tyrosine phosphorylation of c-Met, immunoprecipitation studies were done next to determine whether Np-1 interacted directly with c-Met. Accordingly, lysates from COLO-357 cells that were stably transfected with the HA-tagged Np-1 cDNA were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with either the anti-HA antibody or the anti–c-Met antibody. Np-1 seemed as a single band on the immunoblot (Fig. 7A ), whereas c-Met seemed as a doublet with a major band of 145 kDa and a minor band of 175 kDa (Fig. 7B), which was consistent with its characteristic glycosylation (30). Similarly, immunoprecipitation of endogenous c-Met, followed by immunoblotting with the anti-HA antibody, revealed that Np-1 associated with c-Met (Fig. 7C). Next, lysates from COS7 cells that were transiently transfected with the HA-tagged Np-1 cDNA were subjected to immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti–c-Met antibody. As was the case in the stably transfected COLO-357 cells, endogenous c-Met associated with the transiently transfected Np-1 in COS7 cells (Fig. 7D). By contrast, immunoprecipitation of EGFR (HER1) and the related receptors HER2 and HER3, followed by immunoblotting with the anti-HA antibody, failed to reveal an association between Np-1 and any of these receptors, either in COLO-357 or COS7 (data not shown). Thus, the interaction between Np-1 and c-Met was not limited to pancreatic cancer cells and was relatively specific toward c-Met.


Figure 7
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  		Figure 7. Association of Np-1 with c-Met. A and B, Np-1 immunoprecipitation with anti-HA agarose using lysates from COLO-357 cells expressing the HA-tagged Np-1 in a stable manner. A, immunoblotting was done with the anti-HA (Y-11) antibody, revealing the presence of Np-1. B, immunoblotting with the anti–c-Met antibody (DQ-13) was used to detect c-Met by immunoblotting. C, immunoprecipitation of c-Met with an anti–c-Met (25H2) antibody in parallel lysates prepared from the same COLO-357 cells. The anti-HA (Y-11) antibody was then used to detect Np-1 by immunoblotting. D, COS7 cells. Np-1 was immunoprecipitated with anti-HA agarose, using lysates from COS7 cells that were transiently transfected with the HA-tagged Np-1 cDNA. Immunoblotting was then carried out with the anti–c-Met antibody, indicating that Np-1 associated with c-Met in COS7 cells.

 
Confocal analysis was carried out next to determine the localization of Np-1 and c-Met in COLO-357 cells. This analysis revealed that Np-1 and c-Met are predominantly distributed at the plasma membrane in the absence of ligand, colocalizing in the merged images (Fig. 8, top ). HGF (1 nmol/L) induced the internalization of both Np-1 and c-Met, the fluorescent Np-1 and c-Met signals comigrating from the plasma membrane to the cytoplasm (Fig. 8, middle panels), and, ultimately, to the perinuclear region (Fig. 8, bottom).


Figure 8
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  		Figure 8. Confocal microscopy. COLO-357 cells expressing HA-tagged Np-1 were grown on chamber slides and incubated in the absence (0) or presence (1 nmol/L) of HGF for 30 and 120 min. Cells were then subjected to immunofluorescent staining for HA (green, 12CA5) and c-Met (red, C-12). Nuclei were stained with Hoechst 33258 dye. Regions of colocalization (yellow). Original magnification, 400x.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
c-Met is activated by HGF under physiologic conditions and during embryonic development (31), with HGF expression often limited to cells of mesenchymal origin and exerting its effects on adjoining epithelial cells that express relatively low levels of c-Met (32). By contrast, excessive c-Met activation, as a result of excessive and/or aberrant production of either the receptor or its ligand, has been reported in a number of human malignancies, wherein it has been correlated with enhanced metastatic potential and poor prognosis (33, 34). These deleterious actions occur as a consequence of c-Met–mediated activation of multiple signaling pathways that lead to invasive growth, increased cell proliferation and migration, disruption of intercellular junctions with loss of cell polarity, and epithelial to mesenchymal transformation (26, 27, 35).

Several lines of evidence suggest that c-Met has an important role in PDAC. First, both c-Met and HGF are overexpressed in the cancer cells in PDAC (36). Second, pancreatic cancer cell invasion in vitro is accelerated by HGF (37). Third, NK4, an antagonist of HGF, inhibits pancreatic cancer cell invasion (38). Fourth, insulin-like growth factor-I–mediated invasion of pancreatic cancer cells is dependent on the presence of c-Met (39). Fifth, hypoxia-driven up-regulation of c-Met seems to contribute to the invasive front in PDAC (40). Taken together, these observations suggest that ligation of c-Met by HGF may play a crucial role in the invasive and metastatic potential of pancreatic cancer cells in PDAC.

In the present study, we showed that COLO-357 cells, which express very low levels of endogenous Np-1, were not able to exhibit increased invasiveness in the presence of HGF although these cells are capable of responding to EGF (41) and TGF ß-1 (22) with increased invasiveness. However, stable overexpression of Np-1 in these cells resulted in a marked increase in invasiveness in response to HGF, and this effect was abolished by siRNA-mediated down-regulation of c-Met. Moreover, overexpression of Np-1 in COLO-357 cells markedly enhanced the effects of HGF on c-Met tyrosine phosphorylation. Conversely, in PANC-1 cells, which express relatively high levels of endogenous Np-1, HGF-enhanced invasion, and this effect was completely abolished after suppression of Np-1 expression. Taken together, these observations indicate that Np-1 facilitates c-Met activation by HGF in these cells, thereby leading to stimulation of pancreatic cancer cell invasion.

Overexpression of Np-1 in COLO-357 cells also enhanced HGF-mediated activation of p-38 MAPK. Moreover, SB203580, a specific inhibitor of p38 MAPK, completely suppressed HGF-induced invasion in these cells, indicating that the HGF-mediated increase in invasiveness was due to the Np-1–dependent increase in p38 MAPK activation. In agreement with this conclusion, HGF was previously reported to induce cell migration via p38 MAPK in epithelial cells (42). PP2 and LY294002, which inhibit Src and PI3K, respectively, also suppressed HGF-induced invasion in Np-1–overexpressing cells, implying a role for both pathways in the HGF-mediated increase in invasion in these cells. By contrast, U0126, a MEK inhibitor, did not alter HGF-stimulated invasion in these cells although it is known to do so in some cell types (43). Instead, U0126 enhanced HGF-mediated cell invasion in the sham-transfected cells. The ability of HGF to inhibit ERK activation in the presence of Np-1 while activating p38 MAPK confirms that HGF does not enhance invasion in Np-1–overexpressing cells by activating ERK1/ERK2 and raises the possibility that HGF may activate MAPK phosphatases 3 and/or 4 in the presence of Np-1 because these phosphatases are known to preferentially dephosphorylate ERK (44).

HGF also failed to increase Rac activity in Np-1–overexpressing COLO-357 cells. However, basal Rac activity was markedly increased in these cells by comparison with the sham-transfected cells. Thus, in the presence of high levels of Np-1 that up-regulate Rac, c-Met coordinately activates the p38 MAPK, PI3K, and Src pathways in a manner that markedly enhances cancer cell invasion. This conclusion is supported by the observation that HGF-induced association with Met and subsequent c-Src activation play a critical role in HGF-induced cell motility and anchorage-independent growth of mammary carcinomas (45) and that HGF-induced cell motility is dependent on the PI3K and Rac pathways in Madin-Darby canine kidney epithelial cells (46, 47), Rac being required for the formation of actin-rich membrane ruffles, lamellipodia, that are present at the leading edge of migrating cells.

HGF is a heterodimer consisting of a 69-kDa a-chain that is linked by a disulfide bridge to a 34-kDa b-chain (48). Recent studies have shown that HGF interacts with Np-1 (13), implying possible regulatory and functional associations between the two proteins. The present study points to an additional and novel mechanism by which Np-1 modulates HGF actions by interacting with c-Met. Thus, coimmunoprecipitation studies revealed that Np-1 associated directly with c-Met, and this interaction was relatively specific as it was not observed with EGFR family members. Moreover, analysis by confocal microscopy showed that this association occurred on the plasma membrane, and the addition of HGF promoted the internalization of the Np-1–c-Met complex. The ability of HGF to cause NP-1 internalization provides further support for the concept of a functional association of Np-1 with c-Met, as HGF is known to internalize c-Met (49), and raises the possibility that HGF has the capacity to compete with VEGF-A and semaphorins for Np-1 internalization (50). The interaction between c-Met and Np-1 thus represents an additional mechanism for cross-talk and/or signaling adapter functions between c-Met and membrane receptors, such as was previously reported with respect to {alpha}6ß4 integrin (51), CD44 (52), Semaphorin 4D (53), Plexin B1 (54), ß1 integrin (20), and KAI1 (55). It is likely that Np-1–c-Met interactions are also important in other types of cancers, as it has been recently reported that Np-1 enhances Met activation in glioma cells (56).

PDAC is characterized by the ability of pancreatic cancer cells to invade and metastasize, to resist chemotherapeutic agents, and to elicit a desmoplastic and angiogenic reaction that promotes tumor growth (3). Np-1 is overexpressed in the cancer cells in PDAC (9, 10), and suppression of Np-1 expression in PANC-1 cells has been associated with enhanced chemosensitivity to gemcitabine, whereas increased expression of Np-1 in FG pancreatic cancer cells has been associated with attenuated anoikis (57), raising the possibility that Np-1 may contribute to chemoresistance and apoptosis resistance in PDAC. In view of the fact that HGF and c-met are also overexpressed in PDAC (18), our finding that Np-1 is a coreceptor for c-Met that modulates HGF/c-Met signaling pathway in a manner that enhances HGF-mediated cell invasion suggests that Np-1 overexpression may contribute to cancer spread that metastasis in this malignancy. Therefore, strategies aimed at targeting Np-1 and c-Met, and/or interrupting Np-1–c-Met interactions, may have a therapeutic potential in PDAC.


   Acknowledgments
 
Grant support: USPHS grant CA-102687 (M. Korc).

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.

Received 8/28/07. Accepted 9/ 7/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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