The tyrosine kinase receptor c-Met and its ligand hepatocyte growth factor (HGF) are frequently overexpressed and the tumor suppressor PTEN is often mutated in glioblastoma. Because PTEN can interact with c-Met-dependent signaling, we studied the effects of PTEN on c-Met-induced malignancy and associated molecular events and assessed the potential therapeutic value of combining PTEN restoration approaches with HGF/c-Met inhibition. We studied the effects of c-Met activation on cell proliferation, cell cycle progression, cell migration, cell invasion, and associated molecular events in the settings of restored or inhibited PTEN expression in glioblastoma cells. We also assessed the experimental therapeutic effects of combining anti-HGF/c-Met approaches with PTEN restoration or mTOR inhibition. PTEN significantly inhibited HGF-induced proliferation, cell cycle progression, migration, and invasion of glioblastoma cells. PTEN attenuated HGF-induced changes of signal transduction proteins Akt, GSK-3, JNK, and mTOR as well as cell cycle regulatory proteins p27, cyclin E, and E2F-1. Combining PTEN restoration to PTEN-null glioblastoma cells with c-Met and HGF inhibition additively inhibited tumor cell proliferation and cell cycle progression. Similarly, combining a monoclonal anti-HGF antibody (L2G7) with the mTOR inhibitor rapamycin had additive inhibitory effects on glioblastoma cell proliferation. Systemic in vivo delivery of L2G7 and PTEN restoration as well as systemic in vivo deliveries of L2G7 and rapamycin additively inhibited intracranial glioma xenograft growth. These preclinical studies show for the first time that PTEN loss amplifies c-Met-induced glioblastoma malignancy and suggest that combining anti-HGF/c-Met approaches with PTEN restoration or mTOR inhibition is worth testing in a clinical setting. [Mol Cancer Ther 2009;8(2):376–85]

Glioblastoma is the most common and most deadly human primary brain tumor. Patients with glioblastoma usually die within 1 year of diagnosis. Glioblastoma formation and growth has been associated with multiple molecular dysfunctions. Among these, overexpression of the tyrosine kinase receptor c-Met and its ligand hepatocyte growth factor (HGF) and loss of the tumor suppressor PTEN are thought to play important roles. HGF and c-Met are overexpressed in human gliomas, and expression levels correlate with glioma malignancy and grade (15). Inhibition of HGF or c-Met expression leads to inhibition of in vivo brain tumor formation and growth (6, 7). The loss or mutation of the tumor suppressor PTEN is one of the most common genetic occurrences in glioblastoma. Somatic mutations of PTEN are detected in >40% of glioblastomas and PTEN protein expression is very low or absent in two-thirds of these tumors (810). PTEN can interact with c-Met-dependent signaling at various levels. Such interactions include the antagonistic effect of PTEN on phosphatidylinositol 3-kinase, which plays an essential role in c-Met-dependent cell signaling. Another possible interaction consists in PTEN's ability to bind and dephosphorylate the p52 isoform of Shc, thus inhibiting the recruitment of the Grb2 adaptor and the subsequent activation of the mitogen-activated protein kinase (MAPK) cascade, which is another key mediator of HGF malignant effects (11, 12). Using expression microarrays, we showed previously that PTEN restoration to glioblastoma cells changed c-Met-dependent gene expression in a complex and heterogeneous manner (13). However, the effects of PTEN expression on c-Met-dependent malignancy have not been investigated to date. Understanding these effects is important as it would determine if combining HGF/c-Met inhibition with PTEN restoration strategies or strategies that counteract the effects of PTEN loss such as mTOR inhibition could have therapeutic advantage. It would also help determine if the PTEN status affects the efficacy of emerging clinically applicable anti-HGF/c-Met therapies.

In this study, we investigated the molecular and functional interactions between HGF/c-Met and PTEN in glioblastoma. More specifically, we addressed two major questions: (a) How does PTEN expression or loss alter c-Met-dependent malignancy and associated signaling? and (b) Does combining HGF/c-Met inhibition with PTEN restoration or mTOR inhibition have experimental therapeutic advantage? We found that PTEN expression inhibits c-Met-induced cell proliferation, cell cycle progression, cell migration, and cell invasion. PTEN expression attenuated c-Met-induced protein tyrosine phosphorylation and activation or inactivation of multiple signaling molecules and molecular events known to mediate c-Met functions. Moreover, we found that combining HGF/c-Met inhibition with PTEN restoration or mTOR inhibition, which counteracts some effects of PTEN loss, additively inhibits cell proliferation, cell cycle progression, and in vivo tumor growth. These data show that PTEN loss and c-Met activation additively affect glioma malignancy. The findings also provide a rationale for testing combinations of clinically applicable HGF/c-Met inhibitors with PTEN restoration or clinically applicable mTOR inhibitors in clinical trials.

Cell Culture and Reagents

Human U87 glioblastoma cells were grown in Eagle's MEM (Cellgro Mediatech) containing 10% fetal bovine serum (FBS), 0.15% sodium bicarbonate, 1 mmol/L sodium pyruvate, and 0.1 mol/L nonessential amino acids (Life Technologies). Human A172 glioblastoma cells were grown in DMEM (Cellgro Mediatech) supplemented with 10% FBS and 4.5 g/L glucose. Human SF767 glioblastoma cells (a kind gift from Dr. Russel Pieper, University of California at San Francisco) were grown in MEM (Invitrogen) supplemented with 10% FCS. All cells were grown at 37°C in 5% CO2-95% O2. The mTOR inhibitor rapamycin was purchased from Sigma-Aldrich. The anti-HGF monoclonal antibody L2G7 is a kind gift from Galaxy Biotech. PTEN and control small interfering RNA (siRNA) were purchased from Santa Cruz Biotechnology. Human recombinant HGF is a kind gift from Genentech.

Vectors and Transfections

Adenoviruses encoding wild-type PTEN (Ad-PTEN), anti-HGF U1/ribozymes (Ad-U1/HGF), anti-c-Met U1/ribozymes (Ad-U1/Met), and control adenovirus (Ad-control) were constructed as described previously (7, 13). The cells were infected with adenovirus vectors (multiplicity of infection = 10) for 24 h before treatment with 50 ng/mL HGF. Under these conditions, restored PTEN protein levels are of comparable magnitude with PTEN protein levels in normal human astrocytes (13, 14). For stable PTEN expression, the full-length human PTEN cDNA was subcloned into the mammalian expression vector pcDNA3.1-Zeo (Invitrogen) to generate pcDNA/PTEN. Cells were transfected with pcDNA/PTEN using Fugene transfection reagent (Roche Applied Science) according to the manufacturer's instructions. Transfected cells were selected in zeocin and PTEN expression was verified by immunoblotting.

siRNA-Mediated PTEN Knockdown

PTEN expression was inhibited in SF767 cells expressing wild-type PTEN with PTEN siRNA (Santa Cruz Biotechnology). The cells were transfected with 10 nmol/L PTEN siRNA or scrambled control siRNA using Oligofectamine transfection reagent (Invitrogen) according to the manufacturer's instructions. PTEN protein inhibition was verified by immunoblotting.

Growth Curves

U87 cells (30,000 per well) or A172 cells (40,000 per well) were seeded in 10% FBS medium, transferred to 0.1% FBS medium (for c-Met activation experiments), or 3% FBS medium (for c-Met inhibition experiments) and infected with adenoviruses for 24 h before treatment with 50 ng/mL HGF. The cells were harvested by trypsinization every day for 5 days and counted with a hemocytometer. To assess effects of combining the mTOR inhibitor rapamycin with anti-HGF antibody (L2G7) on cell proliferation, U87 cells (30,000 per well) or A172 cells (40,000 per well) were grown in 10% FBS medium, exposed to 0.1% FBS medium, and treated with 40 nmol/L rapamycin and/or 20 μg/mL L2G7. The cells were counted as described above.

Cell Cycle Analysis

The effects of c-Met and PTEN on cell cycle progression were assessed using propidium iodide flow cytometry as described previously (15). Briefly, U87, A172, or SF767 cells were plated in 10% FBS medium overnight and subsequently exposed to 0.1% FBS medium (for c-Met activation experiments) or 3% FBS medium (for c-Met inhibition experiments) and infected with adenoviruses or transfected with siRNA for 24 h before treatment with or without 50 ng/mL HGF for additional 24 h. The cells were washed with PBS, harvested, and fixed in 70% (v/v) ethanol. The cells were then treated with 20 μg DNase-free RNase and stained with propidium iodide. Cell samples were analyzed on a FACScan (Becton Dickinson).

Invasion Assay

The effects of c-Met and PTEN on cell invasion were assessed using a Transwell invasion assay (BD Biosciences). A cell culture insert was coated with 30 μL collagen type IV (250 μg/mL). U87 or A172 cells (1 × 105) were infected with adenoviruses for 24 h, resuspended in 300 μL of 0.1% FBS medium with or without 50 ng/mL HGF, and placed in the top chamber, and 600 μL of 10% FBS medium were placed in the bottom chamber. After incubation for 6 h at 37°C in 5% CO2, the cells on the top membrane surface were mechanically removed. Cells that migrated to the bottom side of the membrane were fixed and stained with 0.1% crystal violet (Sigma-Aldrich). Photographs were taken and stained cells were counted under a microscope in five randomly chosen fields.

Migration Assay

The effects of c-Met and PTEN on cell migration were assessed by a scratch wound assay. Briefly, cells grown in 6-well plates were infected with adenoviruses for 48 h and the medium was changed to 0.1% FBS overnight. The near-confluent cell monolayers were then carefully scratched using 20 μL sterile pipette tips. Cell debris was removed by washing with PBS. The wounded monolayers were kept in 0.1% FBS medium with or without 50 ng/mL HGF for additional 24 h and photographs were taken under a light microscope.

Immunoprecipitation and Immunoblotting

Immunoprecipitation was used to determine if PTEN binds to c-Met. Briefly, U87 and A172 cells were transfected with the PTEN trapping mutant D92 (a kind gift of Dr. Kenneth Yamada, NIH) or GFP (control). D92 irreversibly binds to its target and has been previously successfully used to determine molecules that bind to PTEN (16). Twenty-four hours post-transfection, the cells were treated with 50 ng/mL HGF for 10 min and subsequently lysed with radioimmunoprecipitation assay buffer (1% Igepal, 0.5% sodium deoxycholate, and 0.1% SDS in PBS). Protein (1 mg) was incubated with 4 μg c-Met antibody (Upstate Biotechnology) or PTEN antibody (Cell Signaling Technologies) for 2 h before incubation with protein A plus G beads (Santa Cruz Biotechnology) overnight at 4°C. The beads were collected by centrifugation, washed five times with lysis buffer, heated to 100°C in Laemmli buffer, and subjected to immunoblotting for PTEN or c-Met, respectively, as described below. Immunoblotting was done as described previously using antibodies specific for phosphotyrosine, phospho-c-Met, phospho-MAPK, MAPK, phospho-Akt, Akt, phospho-GSK-3α/β, and GSK-3α/β (Cell Signaling Technologies), Cdk2, cyclin E, E2F-1, PTEN, and β-actin (Santa Cruz Biotechnology), and p27 (BD Biosciences; refs. 15, 17).

Tumor Formation In vivo

The effects of combining L2G7-mediated HGF inhibition with either PTEN restoration or rapamycin-mediated mTOR inhibition on in vivo tumor growth were tested in an intracranial glioblastoma xenograft model. For combinations of PTEN restoration and L2G7 treatment, stable clones of U87 cells expressing wild-type PTEN or control were generated as described above. The clones (3 × 105 cells) were stereotactically implanted into the brains of immunodeficient mice (n = 10). One week post-tumor implantation, the animals were treated with i.p. injections of 100 μg L2G7 or control twice per week for 3 weeks. For combinations of L2G7 and rapamycin treatments, wild-type U87 cells (3 × 105) were stereotactically implanted into the brains of immunodeficient mice (n = 10). One week post-tumor implantation, the animals were treated with i.p. injections of L2G7 (100 μg twice per week), rapamycin (40 μg three times per week), L2G7 + rapamycin (same doses and schedule as single treatments), or vehicle control for 2 weeks. All animals were euthanized 1 week after the last treatment. The brains were removed, sectioned, and stained with H&E. Maximal tumor cross-sectional area was measured by computer-assisted image analysis.

Statistics

All immunoblots show representative results from at least two experiments. All other experiments were repeated at least three times and the results were expressed as mean ± SE. When appropriate, two group comparisons were analyzed with a t test and multiple group comparisons were analyzed with a Dunnett's test. P values were calculated. P < 0.05 was considered significant.

PTEN Status Affects c-Met-Dependent Glioblastoma Cell Proliferation and Cell Cycle Progression

We found previously that c-Met activation contributes to glioblastoma malignancy by inducing cell proliferation and cell cycle progression (6, 7). To determine how PTEN expression affects these c-Met-induced malignancy variables, we restored PTEN to PTEN-null U87 and A172 glioblastoma cells and inhibited PTEN in wild-type PTEN SF767 glioblastoma cells and studied the effects of c-Met activation on cell proliferation and cell cycle progression in these settings. PTEN was restored to U87 and A172 cells by infection with Ad-PTEN or Ad-Control for 24 h (multiplicity of infection = 10). The cells were subsequently treated with or without 50 ng/mL HGF. Cell proliferation and cell cycle were analyzed as described in Materials and Methods. PTEN expression significantly inhibited basal and HGF-induced cell proliferation in both cell lines (n = 6; P < 0.05, PTEN relative to control and PTEN + HGF relative to control + HGF; Fig. 1A). PTEN expression also significantly and completely inhibited HGF-induced cell cycle progression in U87 cells (n = 3; P < 0.05) and partially inhibited HGF-induced cell cycle progression in A172 cells (n = 4; P < 0.05; Fig. 1B). To assess the effects of endogenous PTEN on c-Met-dependent cell proliferation and cell cycle progression, we inhibited PTEN in wild-type PTEN SF767 cells by transfection with 10 nmol/L PTEN siRNA for 48 h before treatment with or without 50 ng/mL HGF. Inhibition of PTEN expression in SF767 cells amplified HGF-induced cell proliferation and cell cycle progression (n = 3; Fig. 1C). These data show that the PTEN status significantly affects c-Met-dependent glioblastoma cell proliferation and cell cycle progression.

Figure 1.

PTEN status affects c-Met-dependent glioblastoma cell proliferation and cell cycle progression. A and B, PTEN was expressed in PTEN-null U87 and A172 glioblastoma cells via adenovirus (Ad-PTEN) infections before treatment with HGF. The effects of PTEN expression on cell proliferation and G1-S cell cycle progression were assessed by cell counting and by propidium iodide flow cytometry, respectively. The results show that PTEN restoration significantly inhibits basal and HGF-induced cell proliferation in U87 and A172 cells (A). Similarly, PTEN expression inhibited basal and HGF-induced cell cycle progression in U87 and A172 cells (B). *, P < 0.05, relative to Ad-Control; #, P < 0.05, relative to Ad-Control + HGF. Bars, SE. C, PTEN expression was inhibited in wild-type PTEN SF767 cells by siRNA transfection before treatment with HGF. Cell proliferation and cell cycle progression were analyzed as described above. The results show that inhibition of PTEN expression and activation of c-Met additively induce cell proliferation and cell cycle progression. The immunoblots confirm PTEN protein restoration in U87 and A172 and PTEN protein inhibition in SF767 cells. Ad-con, control adenovirus. Bars, SE.

Figure 1.

PTEN status affects c-Met-dependent glioblastoma cell proliferation and cell cycle progression. A and B, PTEN was expressed in PTEN-null U87 and A172 glioblastoma cells via adenovirus (Ad-PTEN) infections before treatment with HGF. The effects of PTEN expression on cell proliferation and G1-S cell cycle progression were assessed by cell counting and by propidium iodide flow cytometry, respectively. The results show that PTEN restoration significantly inhibits basal and HGF-induced cell proliferation in U87 and A172 cells (A). Similarly, PTEN expression inhibited basal and HGF-induced cell cycle progression in U87 and A172 cells (B). *, P < 0.05, relative to Ad-Control; #, P < 0.05, relative to Ad-Control + HGF. Bars, SE. C, PTEN expression was inhibited in wild-type PTEN SF767 cells by siRNA transfection before treatment with HGF. Cell proliferation and cell cycle progression were analyzed as described above. The results show that inhibition of PTEN expression and activation of c-Met additively induce cell proliferation and cell cycle progression. The immunoblots confirm PTEN protein restoration in U87 and A172 and PTEN protein inhibition in SF767 cells. Ad-con, control adenovirus. Bars, SE.

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PTEN Expression Inhibits c-Met-Dependent Glioblastoma Cell Invasion and Cell Migration

c-Met activation contributes to glioblastoma malignancy by inducing tumor cell migration and invasion (1820). To determine if PTEN expression affects c-Met-dependent glioblastoma cell migration and invasion, we restored PTEN to PTEN-null U87 and A172 cells and assessed the effects of c-Met activation on these malignancy variables. PTEN was restored to U87 and A172 cells by infection with Ad-PTEN or Ad-Control for 24 h (multiplicity of infection = 10). The cells were subsequently treated with or without 50 ng/mL HGF and cell invasion and migration were analyzed as described in Materials and Methods. PTEN expression significantly inhibited HGF-induced Transwell cell invasion in U87 cells (n = 3; P < 0.05, PTEN + HGF relative to control + HGF; Fig. 2A). PTEN expression significantly inhibited basal as well as HGF-induced Transwell cell invasion in A172 cells (n = 3; P < 0.05, PTEN relative to control and PTEN + HGF relative to control + HGF; Fig. 2A). PTEN expression also inhibited basal and HGF-induced cell migration in U87 cells (Fig. 2B). These data show that the PTEN status strongly affects c-Met-dependent glioblastoma cell invasion and cell migration.

Figure 2.

PTEN expression inhibits c-Met-dependent glioblastoma cell invasion and cell migration. PTEN was expressed in PTEN-null U87 and A172 glioblastoma cells via adenovirus (Ad-PTEN) infections before treatment with HGF. The effects of PTEN expression on HGF-induced cell invasion and cell migration were analyzed using a Transwell invasion assay and a migration assay. A, PTEN expression inhibited basal and HGF-induced cell invasion in U87 and A172 cells. Photographs show representative stained membranes. Graphs show quantification of invading cells. *, P < 0.05, relative to control; #, P < 0.05, relative to control + HGF. Bars, SE. B, migrating U87 cells show that PTEN expression inhibits basal and HGF-induced tumor cell migration.

Figure 2.

PTEN expression inhibits c-Met-dependent glioblastoma cell invasion and cell migration. PTEN was expressed in PTEN-null U87 and A172 glioblastoma cells via adenovirus (Ad-PTEN) infections before treatment with HGF. The effects of PTEN expression on HGF-induced cell invasion and cell migration were analyzed using a Transwell invasion assay and a migration assay. A, PTEN expression inhibited basal and HGF-induced cell invasion in U87 and A172 cells. Photographs show representative stained membranes. Graphs show quantification of invading cells. *, P < 0.05, relative to control; #, P < 0.05, relative to control + HGF. Bars, SE. B, migrating U87 cells show that PTEN expression inhibits basal and HGF-induced tumor cell migration.

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PTEN Attenuates c-Met-Dependent Signal Transduction

To determine how PTEN expression affects c-Met-dependent signal transduction, we restored PTEN to PTEN-null U87 and A172 cells and assessed the effects of c-Met activation on overall protein tyrosine phosphorylation and on specific signal transduction molecules shown previously to mediate c-Met functions (6, 7, 17, 21, 22). PTEN was restored to U87 and A172 cells by infection with Ad-PTEN or Ad-Control for 24 h (multiplicity of infection = 10). The cells were subsequently treated with or without 50 ng/mL HGF for different times (5 min-24 h). Cell lysates were extracted and immunoblotted with anti-phosphotyrosine or with antibodies specific to the proteins being investigated. HGF treatment induced tyrosine phosphorylation of various proteins in both cell lines. PTEN restoration inhibited basal as well as HGF-induced tyrosine phosphorylation of some of these proteins in U87 cells and HGF-induced tyrosine phosphorylation in A172 cells (Fig. 3A). This suggests that PTEN inhibits c-Met-dependent signaling by inhibiting various molecules that are activated by HGF. To determine if PTEN directly affects c-Met phosphorylation, we used immunoblotting and coimmunoprecipitation and found that PTEN does not alter c-Met phosphorylation relative to total c-Met and does not coimmunoprecipitate with c-Met (data not shown). Therefore, PTEN appears to act on molecules downstream of c-Met. To identify some of the c-Met-activated molecules that are inhibited by PTEN, the effects of PTEN and HGF on selected proteins known to mediate c-Met functions were investigated. The cells were treated as described above and analyzed for protein levels and activation with immunoblotting using specific antibodies and phospho-antibodies. Treatment of the cells with HGF markedly increased the phosphorylation of Akt, p42/44 MAPK, JNK, GSK-3α/β, and mTOR, induced protein levels of Cdk2 and E2F-1, and inhibited protein levels of p27. PTEN expression inhibited HGF-induced phosphorylation of Akt, JNK, GSK-3α/β, and mTOR but did not affect HGF-induced phosphorylation of p42/44 MAPK. Consistent with its effects on the cell cycle, PTEN restoration inhibited HGF-induced E2F-1, partially inhibited HGF-induced Cdk2, and blocked HGF-induced inhibition of p27 (Fig. 3B). These data show that PTEN strongly but selectively affects c-Met-dependent signal transduction.

Figure 3.

PTEN attenuates c-Met-dependent signal transduction. PTEN-null U87 and A172 cells were infected with adenoviruses encoding PTEN (Ad-PTEN) or control (Ad-control) for 24 h before treatment with HGF for either 5 min (Akt), 15 min (MAPK, JNK, GSK, mTOR, and phospho-Tyr), or 24 h (p27, Cdk2, and E2F-1). A, total protein was electrophoretically separated and immunoblotted for phosphotyrosine to determine the effects of PTEN on HGF-induced overall protein tyrosine phosphorylation. The results show that PTEN alters basal and HGF-induced total tyrosine phosphorylation. B, cell lysates were immunoblotted for selected signal transduction proteins know to mediate c-Met functions. The results show that PTEN inhibits HGF-induced phosphorylation of Akt, JNK, GSK, and mTOR and the HGF-mediated changes in p27, Cdk2, and E2F-1 expression but does not affect of the phosphorylation of p42/p44 MAPK.

Figure 3.

PTEN attenuates c-Met-dependent signal transduction. PTEN-null U87 and A172 cells were infected with adenoviruses encoding PTEN (Ad-PTEN) or control (Ad-control) for 24 h before treatment with HGF for either 5 min (Akt), 15 min (MAPK, JNK, GSK, mTOR, and phospho-Tyr), or 24 h (p27, Cdk2, and E2F-1). A, total protein was electrophoretically separated and immunoblotted for phosphotyrosine to determine the effects of PTEN on HGF-induced overall protein tyrosine phosphorylation. The results show that PTEN alters basal and HGF-induced total tyrosine phosphorylation. B, cell lysates were immunoblotted for selected signal transduction proteins know to mediate c-Met functions. The results show that PTEN inhibits HGF-induced phosphorylation of Akt, JNK, GSK, and mTOR and the HGF-mediated changes in p27, Cdk2, and E2F-1 expression but does not affect of the phosphorylation of p42/p44 MAPK.

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Combination of HGF/c-Met Inhibition and PTEN Restoration or mTOR Inhibition Additively Inhibit Glioblastoma Cell Proliferation and Cell Cycle Progression

Because c-Met activation has greater malignant effects in the setting of PTEN loss, we hypothesized that combining PTEN restoration with HGF/c-Met inhibition might lead to greater antiproliferative effects in glioblastoma. To test this hypothesis, we restored PTEN and inhibited HGF/c-Met singly and in combination and assessed the effects on cell proliferation and cell cycle progression. PTEN was restored by infection of U87 cells with Ad-PTEN, and HGF and c-Met were inhibited by infection of the cells with Ad-U1/HGF and Ad-U1/Met, respectively, as described previously (6, 7). PTEN expression and HGF and c-Met knockdown were verified by immunoblotting. Cell proliferation and cell cycle were assessed as described above. Single inhibition of HGF/c-Met as well as single PTEN restoration led to inhibition of cell proliferation and cell cycle progression (Fig. 4A). Combined inhibition of HGF/c-Met and PTEN restoration led to significantly greater inhibitory effects on cell cycle progression (n = 9; P < 0.05 for G0-G1 fraction) and cell proliferation (n = 3; P < 0.05 at day 5) compared with single treatment (Fig. 4A).

Figure 4.

Combined HGF/c-met inhibition and PTEN restoration or mTOR inhibition additively inhibit glioblastoma cell proliferation and cell cycle progression. A, U87 cells were infected with adenoviruses encoding anti-HGF and anti-c-Met U1/ribozymes (Ad-U1/ribozymes), PTEN (Ad-PTEN), a combination of both, or control (Ad-Control). Cell proliferation and cell cycle were assessed by cell counting and by propidium iodide flow cytometry, respectively. The results show that combining PTEN restoration with HGF/c-Met expression inhibition additively inhibits glioblastoma cell proliferation and cell cycle progression. The blots confirm HGF and c-Met knockdown in the cells. *, P < 0.05, relative to control; #, P < 0.05, relative to single treatment. Bars, SE. B, U87 and A172 cells were treated with anti-HGF monoclonal antibody (L2G7), the mTOR inhibitor rapamycin, a combination of both, or solvent control and assessed for cell proliferation as described above. The results show that combining clinically applicable L2G7-mediated inhibition of HGF with clinically applicable rapamycin-mediated inhibition of mTOR additively inhibits glioblastoma cell proliferation. *, P < 0.05, relative to control; #, P < 0.05, relative to single treatment. Bars, SE.

Figure 4.

Combined HGF/c-met inhibition and PTEN restoration or mTOR inhibition additively inhibit glioblastoma cell proliferation and cell cycle progression. A, U87 cells were infected with adenoviruses encoding anti-HGF and anti-c-Met U1/ribozymes (Ad-U1/ribozymes), PTEN (Ad-PTEN), a combination of both, or control (Ad-Control). Cell proliferation and cell cycle were assessed by cell counting and by propidium iodide flow cytometry, respectively. The results show that combining PTEN restoration with HGF/c-Met expression inhibition additively inhibits glioblastoma cell proliferation and cell cycle progression. The blots confirm HGF and c-Met knockdown in the cells. *, P < 0.05, relative to control; #, P < 0.05, relative to single treatment. Bars, SE. B, U87 and A172 cells were treated with anti-HGF monoclonal antibody (L2G7), the mTOR inhibitor rapamycin, a combination of both, or solvent control and assessed for cell proliferation as described above. The results show that combining clinically applicable L2G7-mediated inhibition of HGF with clinically applicable rapamycin-mediated inhibition of mTOR additively inhibits glioblastoma cell proliferation. *, P < 0.05, relative to control; #, P < 0.05, relative to single treatment. Bars, SE.

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Because PTEN gene restoration and U1/ribozyme-mediated HGF/c-Met inhibition are presently not clinically applicable approaches, we also tested the combined effects of monoclonal anti-HGF antibody-mediated inhibition of HGF in combination with mTOR inhibition with rapamycin. A humanized version of the monoclonal anti-HGF antibody (L2G7) has been developed and will enter clinical trials in the near future. mTOR inhibition was chosen as the closest clinically useful approach to counteracting some effects of PTEN loss. U87 and A172 cells were treated with L2G7, rapamycin, a combination of both, or control and assessed for cell proliferation. L2G7 or rapamycin single treatment significantly inhibited cell proliferation relative to control in both cell lines. Combination treatment of L2G7 and rapamycin had significantly greater inhibitory effects on tumor cell proliferation than each therapy mode alone (P < 0.05, relative to single treatment at day 7 for U87 and day 5 for A172). These data provide for the first time a rationale for testing combinations of anti-HGF/c-Met therapies and anti-mTOR therapies in a clinical setting.

Combinations of HGF Inhibition and PTEN Restoration or mTOR Inhibition Additively Inhibit In vivo Glioblastoma Xenograft Growth

To determine if combining PTEN restoration and HGF inhibition have in vivo experimental therapeutic advantage, we generated PTEN-expressing U87 clones by stable transfection with plasmids encoding PTEN and control clones by transfection with plasmids encoding empty vector. PTEN expression was verified by immunoblotting. Two PTEN-expressing clones and two control clones were implanted in immunodeficient mice 1 week before treatment with i.p. L2G7 or control for 3 weeks. One week after the last treatment, the animals were euthanized and tumor maximal cross-sectional areas were measured. Both PTEN restoration and L2G7 treatment led to a significant reduction of tumor cross-sectional areas relative to control (n = 10; P < 0.05, relative to control; Fig. 5A). The combination of PTEN restoration and L2G7 treatment displayed a significantly greater inhibition of tumor growth than each single treatment (n = 10; P < 0.05, relative to single treatment). Notably, in the combination group, except for one animal, which developed a very small tumor, no animal showed signs of tumor growth but only scar tissue around the needle track. Therefore, combination therapy led to an additive inhibitory effect on in vivo tumor growth and tumorigenicity over single therapies. These data show for the first time that combining PTEN restoration with HGF/c-Met inhibition might have therapeutic value.

Figure 5.

Combined HGF inhibition and PTEN restoration or mTOR inhibition additively inhibit in vivo glioblastoma xenograft growth. A, PTEN-expressing U87 clones were generated by stable transfection with plasmids encoding PTEN. Two PTEN-expressing clones and two control clones (3 × 105 cells) were implanted in immunodeficient mice brains (n = 10) 1 wk before systemic treatment with anti-HGF monoclonal antibody L2G7 for 3 wk. The animals were euthanized 1 wk after the last treatment and tumor cross-sectional areas were measured on H&E-stained brain cross-sections with computer-assisted image analysis. The results show that PTEN restoration and HGF inhibition additively inhibit in vivo glioblastoma tumor growth. Immunoblots show PTEN levels in control and PTEN-expressing clones. Top, quantification of tumor cross-sectional areas; bottom, representative brain tumor cross-sections. *, P < 0.05, relative to control; #, P < 0.05, relative to L2G7 and PTEN. Bars, SE. B, wild-type U87 glioblastoma cells (3 × 105) were implanted in immunodeficient mice brains (n = 10). One wk post-tumor implantation, the animals were treated with i.p. injections of L2G7, the mTOR inhibitor rapamycin, L2G7 + rapamycin, or control for 2 wk. The animals were euthanized 1 wk after the last treatment and tumor cross-sectional areas were measured on H&E-stained brain cross-sections with computer-assisted image analysis. The results show that HGF and mTOR inhibitions additively inhibit in vivo glioblastoma tumor growth. *, P < 0.05, relative to control; #, P < 0.05, relative to single treatment. Bars, SE.

Figure 5.

Combined HGF inhibition and PTEN restoration or mTOR inhibition additively inhibit in vivo glioblastoma xenograft growth. A, PTEN-expressing U87 clones were generated by stable transfection with plasmids encoding PTEN. Two PTEN-expressing clones and two control clones (3 × 105 cells) were implanted in immunodeficient mice brains (n = 10) 1 wk before systemic treatment with anti-HGF monoclonal antibody L2G7 for 3 wk. The animals were euthanized 1 wk after the last treatment and tumor cross-sectional areas were measured on H&E-stained brain cross-sections with computer-assisted image analysis. The results show that PTEN restoration and HGF inhibition additively inhibit in vivo glioblastoma tumor growth. Immunoblots show PTEN levels in control and PTEN-expressing clones. Top, quantification of tumor cross-sectional areas; bottom, representative brain tumor cross-sections. *, P < 0.05, relative to control; #, P < 0.05, relative to L2G7 and PTEN. Bars, SE. B, wild-type U87 glioblastoma cells (3 × 105) were implanted in immunodeficient mice brains (n = 10). One wk post-tumor implantation, the animals were treated with i.p. injections of L2G7, the mTOR inhibitor rapamycin, L2G7 + rapamycin, or control for 2 wk. The animals were euthanized 1 wk after the last treatment and tumor cross-sectional areas were measured on H&E-stained brain cross-sections with computer-assisted image analysis. The results show that HGF and mTOR inhibitions additively inhibit in vivo glioblastoma tumor growth. *, P < 0.05, relative to control; #, P < 0.05, relative to single treatment. Bars, SE.

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Because PTEN expression restoration is currently not clinically feasible, we used mTOR inhibition as an alternative approach to counteracting the effects of PTEN loss in combination with L2G7-mediated HGF inhibition. mTOR is activated by PTEN loss and mediates some of the malignant effects of PTEN loss. Also, it was recently shown that mTOR inhibition leads to Akt activation via a feedback mechanism involving receptor tyrosine kinases, providing an additional rationale for combining HGF/c-Met inhibition with mTOR inhibition. We treated established wild-type U87 glioblastoma xenograft-bearing mice (n = 10) with systemic injections of L2G7, the mTOR inhibitor rapamycin, a combination of both, or control for 2 weeks. The animals were euthanized 1 week after the last treatment and tumor sizes were measured. Systemic L2G7 and rapamycin single treatments significantly inhibited tumor growth relative to control (n = 10; P < 0.05 relative to control). Combined systemic delivery of L2G7 and rapamycin led to significantly greater inhibition of xenograft growth than single treatments (n = 10; P < 0.05, relative to single treatment; Fig. 5B). Therefore, combination therapy of L2G7 and rapamycin led to an additive inhibitory effect on in vivo tumor growth over single therapies. These data show for the first time that combining HGF/c-Met inhibition with mTOR inhibition is worth testing in a clinical setting.

Based on its multifunctional and multilevel involvement in human cancer in general and brain tumors in particular, the HGF/c-Met pathway has emerged as a compelling molecular target for therapy. Molecular inhibitors of HGF and c-Met pathway, including neutralizing monoclonal antibodies to HGF and small-molecule inhibitors of c-Met kinase, have been developed (2325). These agents are under active investigation in preparation for future clinical trials. Because PTEN could interact with c-Met-dependent signaling, the success of anti-c-Met/HGF therapies could depend on the PTEN status of the targeted tumors. Such a dependency was shown for EGFR proangiogenic effects and for anti-EGFR clinical therapies where coexpression of PTEN and EGFRvIII in tumors of human giloblastoma patients was significantly associated with clinical response to EGFR kinase inhibitors (26, 27). The present study sought to investigate the effects of PTEN on c-Met-dependent malignancy and associated molecular events and to determine if combining anti-HGF/c-Met therapies with PTEN functional restoration can lead to improved glioblastoma therapies. We show functional and molecular interactions between PTEN and HGF/c-Met in glioblastoma malignancy. We show that PTEN expression/loss strongly affects c-Met-dependent cell cycle progression, cell proliferation, cell invasion, and cell migration. We find that PTEN strongly but selectively attenuates c-Met-dependent signaling. We also show that combining PTEN restoration or counteracting PTEN loss with mTOR inhibitors with HGF/c-Met inhibition additively inhibits the growth of experimental glioblastoma xenografts.

The effects of PTEN on c-Met-dependent malignancy likely occur at multiple levels. PTEN counteracts the effects of phosphatidylinositol 3-kinase on Akt that is activated by c-Met. This is evidenced by the almost complete inhibition by PTEN of c-Met-induced phosphorylation of Akt and downstream molecules including mTOR. PTEN could also theoretically dephosphorylate proteins that are activated by c-Met as has been reported for Shc, a c-Met docking molecule. PTEN also affects c-Met-dependent expression of genes that regulate malignancy as we have reported previously (13). A combination of the above factors is consistent with the strong effects of PTEN on c-Met-dependent malignancy variables that we describe in the present study. Importantly, this study shows for the first time that combining anti-HGF/c-Met therapies and PTEN restoration or mTOR inhibition has additive antitumor effects in glioblastoma. This finding has preclinical significance as clinically applicable anti-HGF/c-Met agents and clinically applicable mTOR inhibitors have been developed. mTOR is a downstream target and important mediator of phosphatidylinositol 3-kinase/Akt and is activated after PTEN loss (28). Inhibition of mTOR reverses some of the effects of PTEN loss (2931). Inhibition of mTOR was shown to synergize with the antiglioma effects of EGFR inhibition (3234). Because genetic restoration of PTEN is not clinically feasible, we selected mTOR inhibition as a clinically applicable approach to counteracting the effects of PTEN loss. mTOR inhibitors are already in clinical trials of glioma patients (35). Additionally, mTOR inhibition has been shown to induce upstream receptor tyrosine kinase signaling leading to Akt activation, providing additional rationale for combining mTOR inhibitors with anti-HGF/c-Met therapies (36). The monoclonal anti-HGF antibody used in the present study was developed by Galaxy Biotech and shown previously to potently inhibit HGF binding to c-Met as well as glioblastoma cell and xenograft growth (23). In the present study, the antibody was used at a dose that results in maximal antitumor effects against orthotopic U87 glioma xenografts as determined by Galaxy Biotech and by us in pilot experiments (23). This indicates that combining an mTOR inhibitor with an anti-HGF antibody might improve the latter's maximal efficacy against gliomas. This antibody has been humanized and is planned for clinical trials in the near future and a functionally similar anti-HGF mAb (AMG102) is currently in clinical trial for recurrent glioblastoma. Our work provides a rationale for the clinical testing of combinations of anti-HGF/c-Met agents and mTOR inhibitors to achieve better therapeutic outcomes. Our studies also suggest that the PTEN status is likely to affect anti-HGF and anti-c-Met therapies.

Because cancer in general and brain tumors in particular are extremely heterogeneous with multiple molecular dysfunctions and numerous histopathologically distinct entities, therapies that target a single pathway such as HGF/c-Met will at best benefit only a subset of patients. It is likely that HGF/c-Met pathway targeting will be most efficacious in combination with other existing and emerging therapies. Basic and translational research has provided a rationale for combining anti-HGF/c-Met therapies with traditional cytotoxic therapies such as radiotherapy and chemotherapy (37). More recent research showed the redundancy of receptor tyrosine kinase pathways in human cancer and suggested that combining multiple receptor tyrosine kinase inhibitors might be an advantageous therapeutic approach (38). This study extends beyond and complements the above studies by investigating the role a tumor suppressor and its potential targeting in combination with receptor tyrosine kinase pathway inhibition.

In conclusion, we show for the first time in human cancer that PTEN expression affects c-Met-dependent malignancy and associated cell signaling and molecular events and that combining anti-HGF/c-Met approaches with PTEN restoration approaches or mTOR inhibition might have therapeutic value in glioblastoma.

No potential conflicts of interest were disclosed.

Grant support: NIH grant RO1 NS045209 (R. Abounader) and University of Virginia Cancer Center.

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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