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Cancer Research 68, 5236, July 1, 2008. doi: 10.1158/0008-5472.CAN-07-5849
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Dissecting and Targeting the Growth Factor–Dependent and Growth Factor–Independent Extracellular Signal-Regulated Kinase Pathway in Human Schwannoma

Sylwia Ammoun1, Christine Flaiz1, Natalia Ristic1, Jennifer Schuldt2 and C. Oliver Hanemann1

1 Clinical Neurobiology, Peninsula College for Medicine and Dentistry, Plymouth, United Kingdom; and 2 Department of Neurology, Zentrum fuer Klinische Forschung, University of Ulm, Ulm, Germany

Requests for reprints: C. Oliver Hanemann, Clinical Neurobiology, Peninsula College for Medicine and Dentistry, The John Bull Building, Tamar Science Park, Research Way, Plymouth PL6 8BU, United Kingdom. Phone: 44-1752-437-418; Fax: 44-1752-517-846; E-mail: oliver.hanemann{at}pms.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Schwannomas are tumors of the nervous system that occur sporadically and in patients with the cancer predisposition syndrome neurofibromatosis type 2 (NF2). Schwannomas and all NF2-related tumors are caused by loss of the tumor suppressor merlin. Using our human in vitro model for schwannoma, we analyzed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT signaling pathways, their upstream growth factor receptors, and their role in schwannoma cell proliferation and adhesion to find new systemic therapies for these tumors that, to date, are very difficult to treat. We show here that human primary schwannoma cells show an enhanced basal Raf/mitogen-activated protein/ERK kinase/ERK1/2 pathway activity compared with healthy Schwann cells. Due to a strong and prolonged activation of platelet-derived growth factor receptor β (PDGFRβ), which is highly overexpressed, ERK1/2 and AKT activation was further increased in schwannoma, leading to increased proliferation. Using specific inhibitors, we discovered that ERK1/2 activation involves the integrin/focal adhesion kinase/Src/Ras signaling cascades and PDGFRβ-mediated ERK1/2 activation is triggered through the phosphatidylinositol 3-kinase/protein kinase C/Src/c-Raf pathway. Due to the complexity of signals leading to schwannoma cell proliferation, potential new therapeutic agents should target several signaling pathways. The PDGFR and c-Raf inhibitor sorafenib (BAY 43-9006; Bayer Pharmaceuticals), currently approved for treatment of advanced renal cell cancer, inhibits both basal and PDGFRβ-mediated ERK1/2 and AKT activity and decreases cell proliferation in human schwannoma cells, suggesting that this drug constitutes a promising tool to treat schwannomas. We conclude that our schwannoma in vitro model can be used to screen for new therapeutic targets in general and that sorafenib is possible candidate for future clinical trials. [Cancer Res 2008;68(13):5236–45]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Schwannomas are common tumors of the nervous system occurring sporadically and in patients with the inherited cancer predisposition syndrome neurofibromatosis type 2 (NF2). Schwannomas are purely derived from Schwann cells and lack the tumor suppressor protein merlin (1). Merlin is structurally related to the ERM family proteins (ezrin, moesin and radixin; refs. 2, 3) that act as linkers between the actin cytoskeleton and the cell membrane (4). Using our in vitro model for human schwannoma, we have previously shown that merlin loss leads to enhanced proliferation and decreased apoptosis (5), increased cell-matrix adhesion (due to the overexpression of integrins; ref. 6), and decreased cell-cell adhesion (7). The RhoGTPases Rac1 and Cdc42 and their downstream effector p21-activated kinase (PAK) are activated in human primary schwannoma cells and have been linked to the aforementioned characteristics of schwannoma cells (8, 9). PAK inhibits merlin in a negative feedback loop (10, 11), and PAK inhibition leads to decreased proliferation rates and ruffling in merlin-deficient cell lines (12). PAK can cross-talk with the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The ERK1/2 signaling pathway is involved in rat Schwann cell dedifferentiation (13), integrin-mediated cell motility, adhesion (14), and proliferation (15). ERK1/2 that is activated in mouse and rat schwannoma cell lines (16) is inhibited by merlin in different cell lines (17). We therefore analyzed ERK1/2 pathway in human primary schwannomas and assessed its role in cell proliferation and adhesion to identify potential therapeutic targets for the treatment of schwannomas. Merlin additionally inhibits the AKT signaling pathway (18, 19), which is important for rat Schwann cell survival (20).

Activation of ERK1/2 and AKT cascades is triggered by surface receptors, such as integrins and receptor tyrosine kinases (21). One candidate is platelet-derived growth factor receptor β (PDGFRβ), wherein internalization and degradation is merlin-dependent in human schwannoma cell line HEI 193 (18). Moreover, PDGFRβ interacts directly with integrins (22), which are overexpressed in human primary schwannoma cells (6). We thus investigated the role of PDGFRβ and other growth factor receptors acting upstream of the ERK1/2 and AKT pathways in human primary schwannoma cells. We show that ERK1/2 and subsequent enhanced proliferation occur through a strong and prolonged activation of PDGFRβ after stimulation of human primary schwannoma cells with the PDGFRβ-specific agonist, platelet-derived growth factor-DD (PDGF-DD). In addition, schwannoma cells display an enhanced basal ERK1/2 activity compared with Schwann cells. Strong AKT activity was also observed in schwannoma cells upon stimulation with PDGF-DD. Using specific inhibitors, we characterized the mechanisms of ERK1/2 and AKT activation. Basal ERK1/2 activation involves the integrin/focal adhesion kinase (FAK)/Src/Ras signaling cascade, and PDGFRβ-mediated ERK1/2 activation involves the phosphatidylinositol 3-kinase (PI3K)/protein kinase C (PKC)/Src/c-Raf pathway. Cross-talk between ERK1/2 and AKT cascades was also observed. Based on our data, we conclude that multiple cascades underlie schwannoma development and therefore suggest that potential therapeutic agents should target several signaling pathways. We therefore tested the PDGFR and c-Raf inhibitor sorafenib (BAY 43-9006; Bayer Pharmaceuticals), currently approved for the treatment of advanced renal cell cancer (RCC; ref. 23). We here show that sorafenib inhibits both basal and PDGFRβ-mediated ERK1/2 and AKT activity and decreases proliferation of human schwannoma cells, suggesting that this drug could be a useful tool in the treatment of schwannomas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Isolation and culture of human Schwann and schwannoma cells. Human Schwann cells from healthy nerve donors and schwannoma cells from patients with NF2 (after informed consent) were isolated, as previously described (24, 25). Diagnosis of NF2 was established by clinical criteria (26). Cell were harvested and resuspended in proliferation medium (GFM): DMEM, 10% FCS, 0.5 µmol/L forskolin, 10 nmol/L β heregulin, 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX), and 2.5 µg/mL insulin, as described in ref. 25.

Chemicals. GF109203X, LY294002, SU6656, U0126 (kindly supplied by Prof. Jyrki Kukkonen, University of Helsinki), IPA-3 (kindly supplied by Dr. Jeffrey R Peterson, Fox Chase Cancer Center), AG1296, 5-bromo-2'-deoxyuridine (BrdUrd) and FTI-277 were from Calbiochem. 12-O-tetradecanoylphorbol-13-acetate (TPA), GW5074, t-octylphenoxypolyethoxyethanol (Triton X-100), polyoxyethylene sorbitan monolaurate (Tween 20), insulin, and IBMX were from Sigma. Wortmannin (kindly supplied by Prof. Jyrki Kukkonen, University of Helsinki) was from Tocris Cookson Ltd., PDGF-DD from R&D Systems. Hoechst 33352 was from Molecular Probes (Eugene), and sorafenib (BAY 43-9006) was kindly supplied by Bayer Pharmaceuticals (West Haven). All inhibitors were added 30 min before PDGF-DD stimulation.

Immunoblotting. Cells were cultured in precoated 35-mm plates to ~70% confluency, serum-starved for 24 h, stimulated, and lysed (5). The immunoblotting was performed, as described in refs. 8, 9. Membranes were incubated with antiactive mitogen-activated protein kinase (MAPK; anti-pThr183-pTyr185-ERK1/2; 1:2,000; Promega), anti–phosphorylated AKT (Ser473; 1:500; Cell Signaling), anti–phosphorylated MAPK/ERK kinase 1/2 (MEK1/2) (Ser298; 1:500; Cell Signaling), anti–phosphorylated MEK1/2 (Ser217/221; 1:500; Cell Signaling), anti–phosphorylated c-Raf (Ser338; 1:500; Cell Signaling), anti–phosphorylated FAK (p-FAK; Y925; 1:500; Cell Signaling), anti–p-FAK (Y861; 1:500; Stressgen), and anti–p-FAK (Y397; 1:200: Chemicon) primary antibodies, followed by incubation with appropriate horseradish peroxidase–conjugated secondary antibodies (Bio-Rad). Enhanced chemiluminescence (Amersham) was used for detection. RhoGDI was used as a loading control, as it has previously been shown not to be regulated in our system (27) in contrast to standard loading controls. Densities of bands were quantified using FluorS-Multi-Imager (Bio-Rad). After stripping and blocking total ERK, MEK, FAK, and AKT levels were determined using anti–total MAPK (ERK1/2, 1:500; Promega), MEK1/2 (1:500; Cell Signaling), FAK (1:500; Upstate), and AKT (1:500; Cell Signaling) antibodies. The expression levels of PDGFRβ, epidermal growth factor (EGF) receptor (EGFR), ErbB2, and ErbB3 were detected using anti-PDGFRβ (1:500) and anti-EGFR (1:500; Cell Signaling), anti-ErbB2 (NeuC18; 1:1000), and anti-ErbB3 (1:1000) antibodies from Santa Cruz Biotechnology.

Ras pull-down. Cells were grown until ~70% confluence, starved for 24 h, stimulated with 100 ng/mL of PDGF-DD for 3 min in the presence or absence of the Ras inhibitor FTI-277 and lysed. Ras pull-down experiments were performed using the EZ-Detect Ras activation kit (Promega) according to manufacturer's protocol. Western blotting was performed, as described above, using an anti-Ras antibody (1:500; Promega).

Immunocytochemistry. Cells were serum starved for 24 h before stimulation, and immunocytochemistry was performed, as described in refs. 8, 9. Anti–active MAPK (anti-pThr183-pTyr185-ERK1/2; 1:2,000; Promega) and anti–p-FAK Y397 antibodies were used for detection of active ERK1/2 and FAK, respectively. Appropriate Cy3 or ALEXA Fluor 488–labeled secondary antibodies were used (1:500; Sigma).

Adhesion assay. Cells were seeded onto precoated 24-well plates for 3 h (28) using different conditions: DMEM, 100 ng/mL PDGF-DD, and GFM. Adhesion assay was performed as described (6).

BrdUrd incorporation and nuclear staining for cell growth. Cells were cultivated on 96-well plates (Nunc) for 72 h, labeled with 10 µmol/L BrdUrd for 24 h, fixed with 4% paraformaldehyde, and incubated with 2 mol/L HCl. Cells were blocked in 2% (w/v) bovine serum albumin and incubated with anti-BrdUrd (1:100; Calbiochem) followed by ALEXA Fluor 488–labeled secondary antibody (1:500; Molecular Probes). Nuclei were stained with 5 µg/mL Hoechst 33342 for 30 min. Dividing cells (BrdUrd positive) and total cell numbers (Hoechst 33342) were manually counted. All inhibitors were added 30 min before stimulation.

Data analysis. Student's two-tailed t test was used for pairwise comparisons and ANOVA, followed by Tukey's post hoc test, was used for multiple comparisons. All the experiments were performed minimum in triplicates, wherein at least three independent batches of cells from different individuals were used. For cell counting, all cells in the well were counted. ns means (not significant) P > 0.05, * indicates P < 0.05; ** indicates P < 0.01, and *** indicates P < 0.001. Significant values are indicated only for the data in which the results are not self-evident. In figures, mean ± SE is given.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Strong Activation of ERK1/2 in Human Primary Schwannoma Cells
Incubation of serum-starved cells with complete medium (GFM) for 10 minutes, 3 hours, and 24 hours produced a higher phosphorylation/activation intensity of ERK1/2 in schwannoma cells compared with Schwann cells (Fig. 1A ). In addition to bands at 42/44 kDa, we also observed weak but specific bands (confirmed by library searches of the recognized epitope using Phospho.ELM screening) at ~300 kDa, which displayed the same pattern of activity as the lower molecular weight bands. The data in Fig. 1A represent total ERK1/2 activity in cells (combined data of 42/44 kDa and 300 kDa bands). Maximal phosphorylation of ERK1/2 occurred in both cell types 10 minutes poststimulation and was 3-fold higher in schwannoma cells than in Schwann cells. Furthermore, we stimulated schwannoma cells with various components present in GFM and observed that serum phosphorylated ERK1/2 to the levels comparable with GFM, whereas insulin, forskolin, IBMX, and β-heregulin evoked only weak to moderate signals even when used at high concentrations (Fig. 1B and C). Stimulation of schwannoma cells with 100 ng/mL PDGF-DD increased levels of phosphorylated ERK1/2 by 7-fold compared with basal, the same magnitude as serum and GFM (Fig. 1B and C). 100 ng/mL of EGF and fibroblast growth factor acidic (FGFa), whose functionality was tested in NIH3T3 and PC12 cells, did not trigger ERK1/2 activation in schwannoma cells (Fig. 1C). In line with this, schwannoma cells show no expression of EGFR (Supplementary Fig. S1). Moreover, Schwann and schwannoma cells express similar levels of ErbB2 and ErbB3 receptors (Fig. 1D) and β heregulin even at high concentrations (100 nmol/L) displayed much lower potency toward ERK1/2 activation than PDGF-DD (100 ng/mL; Fig. 1B and C).


Figure 1
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  		Figure 1. The role of PDGFRβ in schwannoma cell signaling. A, schwannoma cells display strong and long-lasting ERK1/2 activation compared with Schwann cells. Time-response curves for ERK1/2 activation upon GFM (complete medium) stimulation in Schwann (NF2+/+) and schwannoma (NF2–/–) cells. B and C, PDGF-DD is a potent ERK1/2 activator in schwannoma cells. B, ERK1/2 phosphorylation in schwannoma cells after 10 min stimulation with GFM, FCS, insulin, β heregulin, forskolin, IBMX, and PDGF-DD. C, ERK1/2 phosphorylation after 10 min stimulation of schwannoma cells with β heregulin, PDGF-DD, FGFa, and EGF and PC12 and NIH3T3 cells with FGFa and EGF, respectively. D, ErbB2 and ErbB3 are equally expressed in Schwann and schwannoma cells. In all experiments but D, cells were starved for 24 h before the stimulation. The phosphorylated/activated levels of ERK1/2 and receptor expression were detected by Western blot. A, data are normalized to maximum GFM stimulation (100%). B, data are normalized to the basal level (nonstimulated cells). D, data are normalized to NF2+/+. All data are corrected to loading control RhoGDI. In A, the error bars at 0 and 10 min time point for NF2+/+ are not visible because of very low SD.

 
PDGFRβ Activation Leads to Strong and Sustained Activation of MEK1/2 and ERK1/2 in Schwannoma Cells
PDGF-DD (100 ng/mL) induced strong and long-lasting MEK1/2 and ERK1/2 activity in schwannoma cells (Fig. 2A ). As with GFM, in addition to 42/44 kDa bands, specific phosphorylated ERK1/2 (p-ERK1/2) bands were also observed at 300 kDa. The 300-kDa bands were stronger upon PDGF-DD stimulation than upon GFM stimulation and displayed higher ERK1/2 activity in schwannoma cells compared with Schwann cells already 10 minutes after stimulation in contrast to 42/44 kDa bands where the significant difference was seen only after 1 hour (Fig. 2A, compare rows 2 and 3, top). The specificity of 300-kDa bands was estimated by Phospho.ELM screening and by using 2 µmol/L TPA. Despite strong ERK1/2 activity at 42/44 kDa, no band at 300 kDa was observed upon TPA stimulation, suggesting that 300 kDa bands are specific for PDGF-DD stimulation (Fig. 2B). Thus, to show total ERK activity in cells, Fig. 2A (bottom) shows combined data from 42/44 kDa and 300 kDa bands. Maximal MEK1/2/ERK1/2 phosphorylation occurred 10 minutes after stimulation and persisted at least up to 1 hour at 60% to 70% (MEK1/2) and 40% (ERK1/2) of maximal response and declined to basal levels after 24 hours. In comparison, Schwann cells displayed a weaker and only transient activity, which declined to almost basal levels already within 1 hour after stimulation.


Figure 2
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  		Figure 2. Time-response curves for MEK1/2, ERK1/2, and AKT phosphorylation. A and C, schwannoma cells display strong and long-lasting activation of MEK1/2, ERK1/2, and AKT. Schwann (NF2+/+) and schwannoma cells (NF2–/–) were starved for 24 h and stimulated for the time indicated with PDGF-DD (100 ng/mL). MEK1/2 (A, top and middle), ERK1/2 (A, top and bottom), and AKT (C) phosphorylation/activation was assessed by Western blot. A, top and B, upon PDGF-DD stimulation of ERK1/2, specific bands at 300 kDa (in addition to 42/44 kDa) are observed. TPA (2 µmol/L, 10 min) activates ERK1/2 only at the 42/44 kDa level. A (middle and bottom) and C (bottom), the expression of MEK1/2, ERK1/2, and AKT remains unchanged during PDGF-DD stimulation and displays no difference between Schwann and schwannoma cells. Cells were starved for 24 h and stimulated for the time indicated with PDGF-DD (100 ng/mL). The expression of MEK1/2, ERK1/2, and AKT was assessed by Western blot. In A (bottom; p-MEK1/2 and p-ERK1/2) and C (bottom; p-AKT), the data are normalized to the maximum PDGF-DD stimulation (100%), and in A (bottom; total MEK1/2 and total ERK1/2) and C, (bottom; total AKT) the data are normalized to basal NF2+/+. Empty squares (A, middle and bottom and C, bottom), basal levels for 24 h stimulation. All data are corrected to loading control RhoGDI.

 
AKT is Strongly Activated in Schwannoma Cells upon PDGFRβ Stimulation
AKT showed strong and sustained phosphorylation/activation at Ser473 in schwannoma cells upon stimulation with 100 ng/mL PDGF-DD (Fig. 2C) and was observed only at its expected molecular weight size (60 kDa).

Maximal activity was reached 10 minutes poststimulation and remained higher than basal up to 24 hours. Schwann cells in contrast displayed much weaker (maximal response was approximately twice lower than in schwannoma cells) and transient AKT activity, which decreased to basal levels at already 1 hour poststimulation. Total MEK1/2, ERK1/2, and AKT expression remained unchanged during all this conditions, and no difference between schwannoma and Schwann cells was detected (Fig. 2A, middle and bottom and Fig. 2C, bottom).

PDGFRβ is Overexpressed in Schwannoma Cells
PDGFRβ is overexpressed in schwannoma cells (~2-fold) compared with Schwann cells (Fig. 3A ). Also, schwannoma cells display postponed and impaired PDGFRβ degradation as its levels were decreased only by ~35% within 1 hour of stimulation and by ~65% after 24 hours (Fig. 3B) in contrast to Schwann cells where the decrease was more pronounced (~85%; Fig. 3C and D). The decrease in PDGFRβ phosphorylation was slightly faster than its degradation: it decreased by ~50% (from maximum) 1 hour after stimulation and was abolished 24 hours poststimulation (Fig. 3B) following the same pattern as phosphorylated MEK1/2 (p-MEK1/2) and p-ERK1/2 activation course (see Fig. 2C, middle and bottom).


Figure 3
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  		Figure 3. The expression, activation, and degradation of PDGFRβ in Schwann (NF2+/+) and schwannoma (NF2–/–) cells. A, PDGFRβ is overexpressed in schwannoma cells. Schwann and schwannoma cells were cultured in GFM and PDGFRβ expression estimated. B and C, the degradation and inactivation of PDGFRβ is postponed in schwannoma cells. B, schwannoma cells were serum starved for 24 h and stimulated with PDGF-DD (100 ng/mL) for the time indicated, and phosphorylation/activation and expression of PDGFRβ were estimated. C and D, Schwann and schwannoma cells were serum starved for 24 h and stimulated with 100 ng/mL PDGF-DD for 24 h, and the expression of PDGFRβ was estimated. Western blot method was used in all the experiments. A, data are normalized to NF2+/+. B, data are normalized to basal level (nonstimulated cells) for PDGFRβ and to the maximum PDGF-DD stimulation (100%) for phosphorylated PDGFRβ. C, data are normalized to the basal level (nonstimulated cells). Empty triangle in B, basal level for 24 h stimulation. All data are corrected to loading control RhoGDI.

 
A PDGFRβ-Independent Pathway Contributes to ERK1/2 Activation in Schwannoma Cells
In addition to PDGF-DD mediated MEK1/2 and ERK1/2 activation, our data (Fig. 2A; 0 hours) also shows increased basal levels of phosphorylated MEK1/2 and ERK1/2 in schwannoma cells compared with Schwann cells. Because both MEK1/2 and ERK1/2 are equally expressed in Schwann and schwannoma cells (see Fig. 2A), we can exclude MEK1/2 and ERK1/2 overexpression as a reason for enhanced activity and tested the following possibilities: (a) cross-talk of the Rac/PAK pathway with c-Raf/MEK1/2/ERK1/2 and (b) involvement of integrins which are overexpressed and activated in schwannoma cells (6) and have been reported to lead to the activation of ERK1/2 via the FAK/Src/Ras pathway (29).

The Rac/PAK pathway is not involved in the increased basal activity of MEK1/2 and ERK1/2 in schwannoma cells. In basal conditions, c-Raf was only faintly phosphorylated at PAK phosphorylation site (Ser338; Fig. 4A, top ). MEK1/2, however, was phosphorylated at Ser298 (PAK phosphorylation site) in both Schwann and schwannoma cells, but no difference in the phosphorylation intensity was observed between these two cell types (Fig. 4A, middle). Additionally, PAK inhibitor IPA-3 did not decrease basal ERK1/2 activity. The ERK1/2 activity was instead potentiated, which possibly occurred due to cellular stress (Fig. 4A, bottom) as has been observed previously (30). Thus, it is unlikely that PAK contributes to the enhanced basal ERK1/2 activation.


Figure 4
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  		Figure 4. Dissection of the signaling pathway toward basal ERK1/2 activation in schwannoma cells. The following molecules were investigated: PAK (A) Ras (B and C), Src (C), and FAK (D). In all experiments, the cells were serum starved for 24 h, followed by stimulation for 10 min with complete medium (GFM; A) and 100 ng/mL PDGF-DD (D, third panel). Western blot method was used in all experiments but D (fourth panel), Ras pull-down was used in B, and immunocytochemistry in D, fourth panel. A, PAK is not involved in ERK1/2 activation in basal conditions. Schwannoma cells were starved for 24 h and stimulated with GFM for 10 min. The basal- and GFM- stimulated phosphorylation of c-Raf Ser338 (PAK phosphorylation site) and basal levels of phosphorylated MEK1/2 Ser298 (PAK phosphorylation site) in schwannoma cells were estimated by Western blot (A, top and middle). Cells were pretreated with the PAK inhibitor IPA-3 (20 µmol/L) for 10 min and lysed, and the levels of basal ERK1/2 were determined (A, bottom). B, Ras is strongly activated in schwannoma cells in serum-free conditions. Active Ras (Ras-GTP) levels in Schwann (NF2+/+) and schwannoma (NF2–/–) cells were estimated by Ras pull-down and Western blot after the cells were serum starved for 24 h. C, Ras and Src are involved in MEK1/2 activation in basal conditions. Cells were pretreated with farnesyl protein transferase inhibitor FTI-277 (30 µmol/L) and with Src inhibitor SU6656 (4 µmol/L) for 30 min, and the level of phosphorylated MEK1/2 was determined. D, FAK is overexpressed and strongly activated in schwannoma cells. FAK expression in Schwann (NF2+/+) and schwannoma (NF2–/–) cells was estimated by Western blot after the cells were serum starved for 24 h (first panel). FAK is strongly phosphorylated/activated in schwannoma cells (NF2–/–) compared with Schwann cells (NF2+/+). The cells were serum starved for 24 h, and the levels of phosphorylated/activated FAK (Y397, Y861, and Y925) in Schwann and schwannoma cells were estimated by Western blot (second panel). Basal FAK activity is not further regulated by PDGF-DD. The cells were serum starved for 24 h and stimulated for 10 min with 100 ng/mL PDGF-DD. The basal- and PDGF-DD–stimulated levels of p-FAK (Y397, Y861, and Y925) in schwannoma cells were estimated by Western blot (third panel). Phosphorylated ERK1/2 and p-FAK colocalize at focal adhesions in schwannoma cells. The colocalization of p-FAK with phosphorylated ERK1/2 in focal adhesions was performed after cells were serum starved for 24 h. White arrowheads, colocalizing p-ERK1/2 and p-FAK Y397 in the focal adhesions (fourth panel). B and D (first and second panels), data are normalized to NF2+/+. C and D (third panel), data are normalized to the basal (nonstimulated cells). Dotted lines in C, noninhibited basal response and comparisons to noninhibited response (100%). All Western blot data are corrected to loading control RhoGDI.

 
Increased basal MEK1/2 and ERK1/2 activity in schwannoma cells is mediated via the integrin/FAK/src/Ras pathway. We thus looked at the FAK/Src/Ras pathway. Increased Ras-GTP levels, indicative for Ras activity, were detected by Ras pull-down analyses in serum-starved schwannoma cells compared with Schwann cells (Fig. 4B). To couple basal MEK1/2 activity to Ras and Src, we used H-Ras inhibitor farnesyl protein transferase inhibitor FTI-277 and selective Src family kinase inhibitor SU6656 (4 µmol/L). Both inhibitors decreased significantly basal p-MEK1/2 levels, suggesting Ras and Src to be involved in the pathway toward basal MEK1/2 activation (Fig. 4C).

FAK is overexpressed and displays strong basal activity in schwannoma cells. Focal adhesion kinase (FAK) is a tyrosine kinase that is activated after integrin-mediated attachment of the cells to the extracellular matrix and plays an essential role in integrin signaling and tumorgenesis (31). Upon the interaction of integrins with the extracellular matrix, FAK is autophosphorylated at Y397. If further phosphorylated by Src at Y925, FAK is linked to the Ras/Raf/MEK1/2/ERK1/2 pathway (30), and at Y861, its autophosphorylation is enhanced (32).

Overexpression and increased basal activity of FAK were observed in schwannoma cells compared with Schwann cells (Fig. 4D). FAK was strongly phosphorylated at Y397 (Fig. 4D, second, third, and fourth panels), Y861, and Y925 (Fig. 4D, second and third panels) in schwannoma cells. The phosphorylation intensity of FAK at all sites investigated was not affected by PDGF-DD (Fig. 4D, third panel).

To further investigate the relationship between FAK activation, integrins, and basal ERK1/2 activity, we analyzed the cellular localization of p-ERK1/2 and p-FAK using immunofluorescence double labeling. p-ERK1/2 and p-FAK Y397 colocalize at focal adhesions in schwannoma cells (Fig. 4D, fourth panel), suggesting that the basal ERK activation is due to integrin-mediated activation of the FAK/Src/Ras pathway.

PDGFRβ-Mediated ERK1/2 Activation Employs PKC-Src-c-Raf and Involves Two Different Compartments
To define a specific mediator in the PDGFRβ-mediated signaling cascades toward ERK1/2 and AKT activation in schwannoma cells, we performed a dissection of these pathways by using a variety of different specific inhibitors. The specificity of signaling upon PDGF-DD stimulation was confirmed by using the PDGFRβ inhibitor AG1296 (20 and 40 µmol/L; Fig. 5B ).


Figure 5
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  		Figure 5. The dissection of the PDGF-DD–mediated cascades in schwannoma cells. The ERK1/2 signaling pathway was investigated by analyzing the involvement of Ras (A and B); MEK1/2, PKC, PI3K, and Src (B); PDGFRβ and c-Raf (B and D); and PAK (C). The AKT signaling pathway was investigated by analyzing the involvement of MEK1/2, PKC, PI3K, and Src (B) and PDGFRβ and c-Raf (B and D). In all experiments, the cells were serum starved for 24 h before the stimulation with PDGF-DD (100 ng/mL; A–D) or complete medium (GFM; C, first panel) for 10 min (A; 3 min). The detection of phosphorylated/activated proteins was performed by Western blotting in all experiments but C (fourth panel), where immunocytochemistry method was used. In A, Ras pull-down was performed before Western blotting. In B, the cells were pretreated with PDGFβ inhibitor (AG1296), c-Raf inhibitor (GW5074), MEK1/2 inhibitor (U0126), PKC inhibitor (GF109203X), or with PKC activator TPA, PI3K inhibitors (Wortmannin and LY294002), Src inhibitor (SU6656), and farnesyl protein transferase inhibitor (FTI-277) before stimulation. C, coupling of PAK to ERK1/2 cascade was estimated using anti-phosphorylated c-Raf Ser338 (first panel), anti–p-MEK1/2 Ser298 (second panel) antibodies, and PAK inhibitor IPA-3 (20 µmol/L; third and fourth panels). Fourth panel, the cells were stimulated with PDGF-DD (100 ng/mL) for 10 min with and without IPA-3 (20 µmol/L) and stained for p-ERK1/2 (green) and paxillin (a focal adhesion marker, red). White arrowheads, dot-like structures in the cytosol; red arrowheads, focal adhesions (visualized by paxillin staining in red); white arrows, membrane staining. D, cells were pretreated with sorafenib (Bay 43-9006) before the stimulation. In all the experiments, the inhibitors were added 30 min before the stimulation and TPA was added 24 h before the stimulation and IPA3 for 10 min. Dotted lines in B, noninhibited PDGF-DD response and comparisons to noninhibited response (100%). D, data are normalized to the basal levels (nonstimulated cells). All Western blot data are corrected to loading control RhoGDI.

 
We first focused on 42/44 kDa bands. Ras often plays an essential role in the activation of the ERK1/2 via different mediators (33); however, the main pathways are either via direct coupling to c-Raf or via indirect c-Raf activation through the PI3K/phosphatidylinositol 3-phosphate–dependent kinase-1 (PDK1)/PKC cascade (34). The addition of PDGF-DD (100 ng/mL) for 3 minutes did not further enhance Ras activity (Fig. 5A and Supplementary Fig. S2A, compare rows 1 and 2), despite strong ERK1/2 activation at that time point (data not shown). Additionally, FTI-277 (30 µmol/L) did not inhibit PDGF-DD–mediated ERK1/2 activity (Fig. 5B, top, row 9 and Supplementary Fig. S3, compare rows 2 and 3), suggesting that in schwannoma cells Ras is activated independently of PDGFRβ. The proper function of the FTI-277 is shown via Ras pull-down (Supplementary Fig. S2A and B).

We next investigated the involvement of PI3K, which acts either via Ras or via direct coupling to phosphorylated PDGFR (35), resulting in elevated phosphatidylinositol-3,4,5-trisphosphate levels, PKC activation via PDK1, and finally ERK1/2 activation. The nonsubtype-selective PI3K inhibitors wortmannin (300 nmol/L) and LY294002 (10 µmol/L; ref. 36) reduced PDGF-DD–mediated ERK1/2 activation by 40% to 60% (Fig. 5B, top, columns 6 and 7). In the PI3K-mediated cascade toward activation of ERK1/2, PKC can be one of the mediators. We assessed the role of PKC using the inhibitor of the classic and the novel PKC isoenzymes GF109203X (37) and by prolonged exposure of the cells to the PKC activator TPA (38). Both GF109203X (10 µmol/L) and TPA (100 nmol/L) strongly inhibited PDGF-DD–mediated ERK1/2 activation by ~60% and 40%, respectively (Fig. 5B, top, columns 4 and 5).

Src family protein kinases interact with the ERK1/2 pathway via phosphorylation and activation of c-Raf or PKC{delta} (39). Our data show that PDGFRβ activation in schwannoma cells leads to the phosphorylation of c-Raf at the Src phosphorylation sites Y340/341 (data not shown). We thus investigated the involvement of Src in PDGF-DD–mediated ERK1/2 activation using the selective Src family kinase inhibitor SU6656 (4 µmol/L) that strongly inhibited ERK1/2 activity (Fig. 5B, top, column 8).

Additionally, we explored the involvement of c-Raf using the specific inhibitor GW 5074 (5 µmol/L) that decreased ERK1/2 activity by 80% (Fig. 5B, top, column 2). PAK is not involved in PDGFRβ-mediated ERK1/2 activation in schwannoma cells, as PDGF-DD treatment did not lead to phosphorylation of either c-Raf at the PAK phosphorylation site (Ser338) or MEK1/2 at the PAK phosphorylation site (Ser298; Fig. 5C, first and second panels). Moreover PAK inhibitor IPA-3 displayed no effect on PDGF-DD–stimulated activity of MEK1/2 and ERK1/2 (Fig. 5C, third panel, lines 1 and 3) excluding PAK as a mediator of this ERK1/2 cascade activation.

Using the same inhibitors as for investigation of the ERK1/2 pathway, we show that PDGF-DD–mediated AKT activation follows the traditional pathway engaging PI3K since the inhibitors wortmannin (300 nmol/L) and LY294002 (10 µmol/L) strongly inhibited AKT phosphorylation (Ser473; Fig. 5B, bottom, columns 7 and 8).

Thus, PDGFRβ-mediated ERK1/2 activation displayed at the 42/44 kDa level is independent of Ras and PAK but uses PI3K-PKC-Src-c-Raf–dependent pathway and cross-talk with AKT at the level of PI3K.

Despite similarities, such as involvement of c-Raf, PKC, and Src (Fig. 5B, compare top and middle, columns 2, 4, 5, and 8) and bypassing Ras (Fig. 5B, compare top and middle, column 9) the activity of the ERK1/2 pathway at 42/44 kDa and 300 kDa differ regarding the involvement of MEK1/2, PI3K (Fig. 5B, compare top and middle, columns 3, 6, and 7), and PAK (Fig. 5C, third panel, lines 2 and 3). In contrast to ERK1/2 activation at the 42/44 kDa level that depends on PI3K and MEK1/2, the 300-kDa bands are independent from those kinases. The engagement of PAK also differs because PDGF-DD mediated ERK1/2 activity at the 300-kDa level is almost completely inhibited by the PAK inhibitor IPA-3 (Fig. 5C third panel, line 2) whereas this, at 42/44 kDa, was unaffected (Fig. 5C, third panel, line 3). Additional data obtained by immunocytochemistry showed that IPA-3 completely abolished the ERK1/2 activity in focal adhesions and membrane and also eliminated the p-ERK1/2 clustering in the dot-like structures in the cytosol. However, the total ERK1/2 activity in the cytosol was unaffected, thus confirming Western blot data and suggesting PAK acting as a scaffold in a complex containing ERK (Fig. 5C, fourth panel). Thus, our data indicate that PDGF-DD–mediated ERK1/2 activation at the 300-kDa level engages c-Raf, PKC, src, and PAK and localizes to the different cellular compartments than active ERK1/2 observed at the 42/44 kDa level.

In summary, we found two major mechanisms converging toward ERK1/2 activation in schwannoma cells: (a) basal activation via integrins/src/FAK/Ras and (b) PDGFRβ-mediated via the PI3K-PKC-c-Raf and PKC-PAK-c-Raf pathways were the first mentioned cross talk with and strongly activate AKT (Fig. 6D ).


Figure 6
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  		Figure 6. Signaling pathways involved in the enhanced proliferation of schwannoma cells. A, Schwann (NF2+/+) and schwannoma cells(NF2–/–) were cultured for 72 h with or without PDGF-DD. Total cell amount was estimated by nuclear staining with 5 µg/mL Hoechst 33342 and cell counting. B and C, schwannoma cells were incubated with U0126 (MEK1/2 inhibitor), AG1296 (PDGFRβ inhibitor), and U0126 together with AG1296 (B) and sorafenib (PDGFRβ and Raf inhibitor; C) with and without PDGF-DD for 72 h. BrdUrd (10 µmol/L) was added during the last 24 h of the experiment. The amount of proliferating cells (BrdUrd-positive) was assessed by cell counting. In all experiments, the data are normalized to the basal level (nonstimulated cells). D, signaling pathways targeted by sorafenib in schwannoma cells. Merlin deficiency leads to the activation of Ras, overexpression and activation of integrins, and overexpression and postponed degradation of PDGFRβ. Ras and integrins trigger basal ERK1/2 activity via FAK, which is strongly overexpressed and phosphorylated/activated by Src. Basal ERK1/2 activity is further potentiated via PDGFRβ-mediated PI3K/PKC/Src/c-Raf/MEK1/2 and cascade. PAK acts as a scaffold in a complex containing active ERK1/2.

 
We thus chose sorafenib (1 and 10 µmol/L), an inhibitor of PDGFR and c-Raf (Fig. 6D) already used in clinic, which efficiently inhibited both basal- and PDGF-DD–stimulated ERK1/2 and AKT activity (Fig. 5D), suggesting that this compound has a potential therapeutic effect on schwannoma.

PDGFRβ Induces Enhanced Cell Proliferation, but not Adhesion, via ERK1/2 in Schwannoma Cells
We next assessed the functional role of PDGFRβ-mediated ERK1/2 activation in schwannoma cells.

Adhesion. First, we performed an adhesion assay (6) where cells were cultured in three different conditions: DMEM only, DMEM with 100 ng/mL PDGF-DD, and complete medium (GFM). Only GFM but not PDFG-DD alone induced schwannoma cell adhesion (data not shown); thus, it is unlikely that PDGFRβ alone play a role in this process.

Proliferation. We have previously shown that schwannoma cells display an increased proliferation rate compared with Schwann cells (40). As PDGF is a potent growth factor in several cell types, we tested its mitogenic potency in our in vitro model. The addition of 100 ng/mL PDGF-DD for 72 hours resulted in an increase of total cell number (determined by Hoechst 33342 staining of the nucleus) by ~40% in schwannoma cells, whereas no mitogenic effect on Schwann cells was detected (Fig. 6A) under our culture conditions. Additional experiments measuring BrdUrd incorporation revealed that schwannoma cells displayed basal proliferation activity, which was strongly potentiated by PDGF-DD by ~80% (Fig. 6B and C), indicating that PDGF-DD is a potent mitogen for human primary schwannoma cells. Both MEK1/2 and PDGFR inhibitors U0126 (1 µmol/L) and AG1296 (20 µmol/L), respectively, strongly reduced PDGF-DD–mediated as well basal cell proliferation (Fig. 6B), suggesting that the ERK1/2 pathway is involved in PDGFRβ-mediated proliferation in schwannoma cells. Next, we tested sorafenib (0.1, 0.5, and 1 µmol/L), a PDGFR and c-Raf inhibitor that is currently approved for clinical use in advanced RCC. Interestingly, 0.5 µmol/L sorafenib effectively inhibited PDGF-DD but not basal and 1 µmol/L inhibited both PDGF-DD–mediated and basal proliferation in schwannoma cells (Fig. 6C), suggesting that this component could be used for schwannoma treatment.


   Discussion
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We show strong and prolonged ERK1/2 pathway activation in human primary schwannoma cells compared with primary Schwann cells. PDGFRβ is strongly overexpressed in human primary schwannoma cells in agreement with a previous observation in human schwannoma tissue (18). Insulin and β heregulin activate ERK1/2 only weakly, even when used at high concentrations. FGFa and EGF fail to activate ERK1/2. Additionally, the expression levels of ErbB2 and ErbB3 receptors are equal in human schwannoma and Schwann cells. EGFR is present in the rat schwannoma cell line RT4 (data not shown), but not in human primary Schwann and schwannoma cells. Thus, PDGFRβ is a crucial receptor signaling through ERK1/2 pathway in human schwannoma.

In addition to the activation of the ERK1/2 pathway, PDGF-DD also evokes a strong and prolonged AKT response in schwannoma cells compared with Schwann cells. Although PDGFRβ is important for the activation of the AKT pathway, as shown here, we cannot exclude that, in different physiologic milieu, other growth factors could also be involved in activation of this pathway.

PDGFRβ displays a postponed degradation in schwannoma cells compared with Schwann cells, in agreement with a previous study on human schwannoma cell line HEI193 (18). The receptor activity is prolonged and, 1 hour after stimulation, decreased only by 50% of maximum response. The kinetics of PDGFR deactivation is usually much faster, as shown in fibroblasts, where already 20 minutes after stimulation the activity dropped by 75% (41). Our data suggest that one of functions of merlin is to regulate the receptor activity. PDGFRβ phosphorylation/activity display the same pattern as MEK1/2 and ERK1/2 activity, but diverge from AKT, which exhibit strong activity even 24 hours after stimulation. Thus, in schwannoma cells, AKT activation is regulated not only at the PDGFRβ level but possibly also by positive feedback mechanisms downstream of the receptor, e.g., saturation of PI3K activity (41), prolongation of signaling in endosomes (42), or activation of phosphatase inhibitors (43). These human data extend the observations made in the rat schwannoma cell line RT4 and in a mouse NF2–/– Schwann cell line, where receptor expression and function were not affected by merlin (16). In addition to strong and long-lasting PDGF-DD–mediated MEK1/2 and ERK1/2 activity, schwannoma cells display stronger basal activity of these kinases compared with Schwann cells. This could involve either Rac/PAK or integrin-mediated pathways. PAK can be excluded as an activator of ERK1/2 at 42/44kDa level, which is the main ERK1/2 band in basal conditions, as c-Raf is not phosphorylated at its PAK phosphorylation site (Ser338) and analysis of the PAK-dependent phosphorylation site on MEK1/2 (Ser298) shows that schwannoma and Schwann cells display similar phosphorylation levels. Also, the PAK inhibitor IPA-3 inhibited neither basal- nor PDGF-DD–stimulated MEK1/2 and ERK1/2 activity. This is an unexpected finding because the Rac1/PAK pathway is strongly up-regulated in schwannoma cells (8) and PAK is known to act as a scaffold to enhance ERK1/2 activity (44). However, we found instead that ERK1/2 activity at the 300-kDa level, prominent after PDGF stimulation, is dependent from PAK that likely acts as a scaffold. Moreover, the activation of the ERK1/2 at the 300-kDa level displays also other differences from this at 42/44 kDa, as it bypasses PI3K and MEK1/2. A potential explanation could be hidden binding sites for those inhibitors caused by complex formation. Immunocytochemistry also show the differential distribution of p-ERK1/2 in schwannoma cells (nucleus, cytosol, membrane, and focal adhesions). Our data thus suggest that PDGF-DD mediates the activation of ERK1/2 in different cellular compartments which display different molecular background. We next tested the role of integrins that are up-regulated in schwannoma (6) and may activate ERK1/2 via the FAK/src/Ras pathway (29). We detected enhanced basal activity of Ras, MEK1/2, and ERK1/2 and increased expression and activation of FAK in schwannoma cells. Additionally, p-FAK colocalizes with p-ERK1/2 in focal adhesions. Moreover, blocking of Ras and Src inhibit basal MEK1/2 activity. Taken together, these results indicate that integrins are likely to contribute to ERK1/2 activation in schwannoma cells via the FAK/src/Ras pathway under basal conditions. We think that in basal conditions schwannoma cells display activation of the integrin/FAK/Src cascade, as well as increased levels of active Ras, which, upon coupling to p-FAK (Y925), leads to activation of ERK1/2.

As the stimulation of PDGFRβ strongly potentiates ERK1/2 and AKT activity, we consider that this receptor and its downstream signaling pathways is important for schwannoma development. To define a potential therapeutic target for schwannoma treatment, we performed a dissection of the cascades downstream of PDGFRβ in addition to basal activation. Our data suggest cooperation between PI3K, PKC, and Src in PDGF-DD–mediated ERK1/2 activation. Although Ras is strongly activated in schwannoma cells and implicated in basal signaling toward ERK1/2 activation, it is not further activated by PDGF-DD and is not involved in the PDGFRβ-mediated pathway. This could be explained by a possible uncoupling of Ras from the receptor. Different Ras isoforms localize to distinct microdomains within plasma membrane and different intracellular compartments leading to diverse signal outputs despite interaction with similar activators and effectors (45). Ras insensitivity to PDGF-DD could also be explained by either saturated basal activity of Ras or a negative feedback from basal ERK1/2 to Grb2-Sos (Ras-GEF; ref. 46). Our data somehow contradicts previous findings, showing Ras involved in PDGFRβ-mediated ERK1/2 activation (16). This discrepancy may be down to the use of rat schwannoma cell line caused by different mutation and displaying malignant properties by Morrison and colleagues, whereas we focused our study on primary human schwannoma.

We show that PDGF-DD engages different pathways to activate ERK1/2 that is localized to different intracellular compartments and detected at different molecular weight levels by Western blot. The p-ERK1/2 pathway at 42/44 kDa (cytosol) favors a model in which c-Raf is activated by Src and PKC, where the later is activated by PI3K (via PDK1), which directly binds to phosphorylated/activated PDGFRβ (Fig. 6D; refs. 47, 48). PI3K acts also as a convergence point to the activation of AKT (Fig. 6D). The p-ERK1/2 pathway at 300-kDa level (focal adhesions) engages c-Raf, PKC, Src, and PAK. Our data thus fit to previous observations showing that ERK1/2 activity is tightly regulated by merlin (17) and involved in rat Schwann cells dedifferentiation (13) and integrin-mediated cell motility and adhesion (14), which is dramatically disregulated in merlin-deficient schwannoma cells (6).

Whereas transient ERK1/2 activity is important for normal cell function, a strong and long-lasting ERK1/2 activation can be mitogenic (49, 50). We show increased proliferation of schwannoma cells upon stimulation with PDGF-DD, whereas no effects were observed in Schwann cells. This could be explained either by the short and weak ERK1/2 activity in those cells or a requirement for additional factors in the culture medium, such as insulin-like growth factors (51). Inhibition of MEK1/2 decreases PDGF-DD–mediated proliferation to basal levels, suggesting that ERK1/2 pathway is involved in this process. Additionally, both MEK1/2 and PDGFRβ inhibition significantly inhibited basal cell proliferation, suggesting that both the integrin/FAK/src/Ras cascade and PDGFRβ-mediated signaling, possibly via autocrine mechanisms, are involved in schwannoma cell proliferation.

Based on this evidence, we consider PDGFRβ-mediated signaling to be involved in schwannoma development. Additionally, the integrin-mediated FAK/src/Ras cascade contributes to basal ERK1/2 activity, thereby potentiating the PDGFRβ-specific pathway (Fig. 6D; ref. 16).

Because different pathways are involved in schwannoma cell proliferation, we suggest that a combined therapy targeting different proteins might be appropriate for treating schwannoma. Promising therapeutic agent include sorafenib, a PDGFR and Raf inhibitor already approved for use in patients with advanced RCC, as it inhibits efficiently both basal and PDGF-DD–mediated ERK1/2 and AKT activation and proliferation in human primary schwannoma cells.


   Disclosure of Potential Conflicts of Interest
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 Abstract
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 Disclosure of Potential...
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No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Children's Tumor Foundation and Peninsula College of Medicine and Dentistry.

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.

We thank Prof. Jyrki Kukkonen (University of Helsinki) for GF109203X, LY294002, SU6656, U0126, and wortmannin, Bayer Pharmaceuticals for sorafenib (BAY 43-9006), and Dr. Jeffrey R Peterson (Fox Chase Cancer Center) for IPA-3.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 10/11/07. Revised 3/ 4/08. Accepted 4/ 9/08.


   References
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 Materials and Methods
 Results
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 Disclosure of Potential...
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