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Cancer Research 68, 1862, March 15, 2008. doi: 10.1158/0008-5472.CAN-07-1182
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

The Protein Phosphatase Activity of PTEN Regulates Src Family Kinases and Controls Glioma Migration

Nandini Dey1, Hal E. Crosswell2, Pradip De1, Ramon Parsons3, Qiong Peng1, Jing Dong Su4 and Donald L. Durden1

1 Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia; 2 Pediatric Hematology and Oncology Center, Greenville Hospital System University Medical Center, Greenville, South Carolina; 3 Institute of Cancer Genetics, College of Physicians and Surgeons, Columbia University, New York, New York; and 4 Semafore Pharmaceuticals, Inc., Indianapolis, Indiana

Requests for reprints: Donald L. Durden, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322. Phone: 404-778-5118; E-mail: don_durden{at}oz.ped.emory.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is mutated or lost in 60% to 70% of advanced gliomas and is associated with malignant phenotypic changes such as migration, which contribute to the morbidity and mortality of this disease. Most of the tumor suppressor function of PTEN has been attributed to its ability to dephosphorylate the second messenger, phosphatidylinositol 3,4,5-triphosphate, resulting in the biological control of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Despite recent work suggesting that the protein phosphatase activity of PTEN controls glioma cell migration, the mechanisms by which this occurs are unclear. Herein, we show using glioma cell lines (U87MG and U373MG) stably transfected with wild-type PTEN or catalytically altered mutants of PTEN that PTEN controls integrin-directed migration in a lipid phosphatase, PI3K/AKT–independent manner. Confirming this observation, we show that the stable overexpression of COOH-terminal Src kinase, the physiologic negative regulator of SRC family kinases (SFK), or treatment with the SFK inhibitor PP1 abrogates glioma migration. The results provide direct evidence that the downstream effect of the protein phosphatase activity of PTEN is to suppress SFK and FYN, and to regulate RAC-GTPase activity after {alpha}v integrin stimulation. Furthermore, studying vitronectin-directed migration using (a) Fyn small interfering RNA and (b) astrocytes from Fyn heterozygous (+/–) mice, Pten heterozygous (+/–) mice, Pten and Fyn double heterozygous (+/–) mice, or Fyn knockout (–/–) mice confirmed a role of FYN in {alpha}v integrin–mediated haptotaxis in glial cells. Our combined results provide direct biochemical and genetic evidence that PTEN's protein phosphatase activity controls FYN kinase function in glioma cells and regulates migration in a PI3K/AKT–independent manner. [Cancer Res 2008;68(6):1862–71]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mutations in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) occur in one third of advanced gliomas and have been shown to correlate with a worse prognosis (1). In other advanced cancers as well as glioblastoma multiforme (GBM), epigenetic events such as promoter methylation, COOH-terminal phosphorylation, oxidation events, and other poorly understood protein modifications may result in loss of PTEN function, contributing to the many downstream effects that occur when this tumor suppressor is lost (2, 3).

Functionally, PTEN is one of three phosphatases known to dephosphorylate the D3 position of phosphatidylinositols, resulting in a down-regulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, and is thus a competitive and potent antagonist to PI3K (4, 5). Interestingly, the initial function of PTEN as a dual-specificity protein phosphatase was discovered based on its protein structure (6, 7); however, its ability to dephosphorylate both tyrosine and serine/threonine sites of acidic peptides has received relatively little attention with regard to its tumor suppressive activity. More attention has been focused on its capacity to antagonize the growth and proliferation-promoting actions of PI3K and AKT via its catalytic activity toward anionic lipid substrates like phosphatidylinositol 3,4,5-triphosphate (PIP3; refs. 4, 6, 8).

More recently, the protein phosphatase activity of PTEN has received increased scrutiny in aberrant cellular processes, particularly regarding cell motility, invasion, migration, and in the nucleus (9–12). Several lines of evidence point to the ability of PTEN to regulate cell motility through protein dephosphorylation. First, the NH2-terminal domain of PTEN has extensive homology with tensin, a protein that interacts with actin filaments at focal adhesion contacts (13). Second, mouse models have revealed that the embryonic lethality associated with homozygous deletion of PTEN and the abnormal central nervous system developments in conditional knockout mouse are largely due to defects in cellular migration (14–17). Also, one PTEN mutation found in Cowden syndrome and associated with a transition of nucleotide 386, which results in a glycine to glutamic acid switch at codon 129 (G129E), maintains protein phosphatase function while having little lipid phosphatase activity (5, 18). The use of this mutation in several in vitro studies has provided indirect evidence for the protein phosphatase activity of PTEN. The mechanism by which PTEN exerts its inhibitory effect on migratory phenotypes, and its specific in vivo protein substrates, have not been fully explored; however, downstream targets of the protein phosphatase activity of PTEN may include mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), cyclin D1, and Shc (9, 10, 13, 19–21).

SRC family kinases (SFK) are non–receptor tyrosine kinases, of which there are 10 members, and are strongly implicated in a wide number of cancers (22, 23). SFKs are overexpressed in many different cancers and are typically associated with advanced malignancies and/or metastatic spread. In addition, SFKs have important functions specific to the motility of cells and are necessary for the growth factor and integrin-stimulated migration of cells in neural development and various malignancies (23, 24). Previously, FYN, LYN, YES, HCK, and SRC have all been shown to be present in neural tissue (25, 26). Specifically, loss of Fyn in mice [Fyn (–/–) mice die perinatally] has been associated with abnormal neural development, suggesting an integral role of SFKs in neural development. Although SRC is known to regulate cell cycle progression and survival through the PI3K/AKT pathway (27, 28), SFK's control of integrin-stimulated adhesion and migration seems to be mediated through FAK and/or MAPK, resulting in rearrangements of cortical actin and cytoskeletal machinery (23, 29). One of the main downstream targets of the cooperativity of FAK and SRC is the small Rho GTPase, RAC. RAC has been shown to control cell motility (29, 30), affecting actin reorganization at the leading edges of cells (31).

Recent evidence suggests that SRC may alter the function of PTEN to regulate the PI3K signaling cascade (32) and may affect PTEN stability, thereby potentiating its growth-enhancing effects. SRC has been shown to increase COOH-terminal tyrosine phosphorylation of PTEN, resulting in regulation of its tumor suppressor function (33). SFKs have been reported to cooperate with receptor tyrosine kinases, culminating in signaling through PTEN/PI3K (34).

Currently, there are no reports of PTEN's regulation of SFKs. Herein, our results show that PTEN regulates glioma cell migration via its protein phosphatase activity independent of lipid phosphatase activity, and suggest that this effect is potentially mediated through its control of FYN and RAC GTPase downstream of {alpha}vβ3 integrin engagement.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Constructs and reagents. Wild-type (WT) PTEN and its mutant (G129E, lipid phosphatase dead or R130M, kinase dead) cDNAs were subcloned into a retroviral expression vector containing a muristirone-inducible promoter or into the pBABEpuro vector for expression in U87MG and U373MG glioma cells (15). WT COOH-terminal Src kinase (CSK) was overexpressed in U87MG cells using retroviral constructs (pLXSN) as previously described (35). U87MG and U373MG cells were maintained in RPMI (Invitrogen), 10% fetal bovine serum (FBS; Hyclone), and 1% penicillin/streptomycin. Brain astrocyte cultures were established from WT mice, Fyn heterozygous (+/–) mice, Pten heterozygous (+/–) mice, Pten and Fyn double heterozygous (+/–) mice, or Fyn knockout mice. Rabbit polyclonal antibodies against phospho-AKT (Ser473), AKT, and PTEN were purchased from Cell Signaling Technology. Antibodies against phospho-SRC (Y418), FYN, SRC, and CSK are purchased from Santa Cruz Biotechnology. FYN immunokinase assay was carried out using a SRC assay kit from Upstate Biotechnology. FYN expression was knocked down using small interfering RNA (siRNA) SMARTpool specific for FYN (Upstate Biotechnology) along with a corresponding negative control. Lipofectamine 2000 was bought from Invitrogen. Vitronectin was purchased from Sigma. Horseradish peroxidase–tagged anti-rabbit IgG and anti-mouse IgG were obtained from Amersham Life Sciences. Goat anti-mouse and anti-rabbit IgG (H+L)–AP (human adsorbed) were purchased from Southern Biotechnology, Inc. PAK-1 PBD agarose (RAC1 assay reagent) for the pull down of activated RAC1 and monoclonal RAC1 antibody were from Upstate Biotechnology. For PTEN phosphatase assay (both lipid and protein phosphatase assays), malachite green solution A and B were bought from Upstate Biotechnology. Lipid substrates [PI(3,4,5) P3DiC8] and protein substrates (YEEEEEpYEEEEEY) were procured from Echelon Biosciences, Inc., and Emory University (Chemistry Division), respectively.

Animals. Mice heterozygous for Pten (+/–) on the C57BL6 genetic background were obtained from Dr. Ramon Parsons (Columbia University, New York, NY) and maintained in the Animal Facility Core at Emory University under an Institutional Animal Care and Use Committee–approved protocol. Genotyping of Fyn knockout mice on the C57BL6 background was performed as described elsewhere (36).

Phosphatase assay. The phosphatase activity of PTEN was determined by malachite green assays. The lipid phosphatase activity of PTEN was measured using water soluble D-myo-PtdIns(3,4,5) P3 (Echelon Bioscience) as a substrate, whereas its protein phosphatase activity was measured using a custom-made acidic peptide YEEEEEpYEEEEEY (Microchemical Core, School of Medicine, Emory University). Reaction mixtures containing 1 µg of glutathione S-transferase (GST) PTEN fusion proteins (purified in the laboratory) and 70 µmol/L D-myo-PtdIns(3,4,5) P3 or 400 µmol/L acidic peptide (YEEEEEpYEEEEEY) were incubated for 40 min at 37°C. The color developed by malachite green (15 min at room temperature) was measured at 650 nm. The amount (pmol) of free phosphate released in each enzyme reaction was determined by linear regression analysis against a standard phosphate curve.

Astrocytes culture. The brain tissue from P0 to P1 mouse pups (dissected on ice) was subjected to trypsin (0.25%) digestion for 5 to 7 min at 37°C. Digested tissue was triturated in presence of DNase I (Invitrogen). The resulting cells were grown in 10% DMEM with 1% penicillin and streptomycin at 37°C in a humidified atmosphere of 5% CO2 in air. Testing for astrocytic markers included glial fibrillary acidic protein (GFAP) by immunohistochemical analysis (>95% GFAP positive at passage 4).

Migration assays. Haptotaxis assays were carried out using transwell migration chambers (Costar Corp.) as previously described (29, 30). Cells (2 x 105 per well) were added into the upper chamber of the transwell containing the membrane (with 8-µm pore) through which they were allowed to migrate over time to the vitronectin-coated (10 µg/mL for 1 h) side. Control experiments involved coating both sides of the membrane with vitronectin. An adhesion assay on vitronectin was performed simultaneously with the haptotaxis assay under similar conditions as described (29). In vitro wound healing assays were performed as previously described (12). In brief, after coating plates with vitronectin (10 µg/mL), wounds were created by scratching the confluent monolayer of cells. The number of cells that migrated into the "scratched" area was counted from randomly chosen fields using either Olympus DP70 system or Axiovert 200 M, Zeiss system. Student's t test was used to determine the statistical significance.

Biochemical analyses. Immunoblots were performed on cell lysates obtained from U87MG and U373MG cells grown in tissue culture (35). Equivalent amounts of protein (Bradford assay) were resolved by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were probed with antisera specific for PTEN, AKT, phospho-S473-AKT, CSK, FYN, phospho-Y418-FYN, RAC1, and β-actin.

In vitro FYN kinase assay. FYN kinase assays were performed using the SRC assay kit from Upstate Biotechnology following the manufacturer's instructions as described elsewhere (35, 37). Cells were stimulated in 10-cm nontissue culture Petri dishes (coated with 10 µg/mL of vitronectin in PBS for 1 h at 37°C). For FYN kinase assay, clarified lysates were immunoprecipitated with FYN antibody bound to protein-G agarose (Santa Cruz Biotechnology). Immunoprecipitates were used for the kinase assay.

Integrin-induced RAC1 activation. Levels of activated RAC1 were determined by the pull-down assay as described elsewhere (29, 37). U87MG cells were stimulated in 10-cm Petri dishes (non–tissue culture) that were coated with 10 µg/mL vitronectin. Clarified lysates were used for the RAC1 pull-down assay.

In vivo knockdown of FYN protein by siRNA. A cell density of 70% to 80% was used for the transfection (Lipofectamine 2000) of Fyn-specific siRNA into U87MG cells. Transfected cells were collected after 24 and 48 h for analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTEN controls vitronectin-mediated migration through its protein phosphatase activity. U87MG and U373MG cells, which are null for PTEN (38, 39), were stably transfected with either fully active PTEN (WT), a catalytically dead mutant of PTEN (R130M), or a protein phosphatase–active, lipid phosphatase–inactive mutant of PTEN (G129E), as described elsewhere (5). We examined the effects of expression of WT PTEN and its different mutants on the downstream activation of AKT in these cells (Figs. 1 and 2 ). As expected, high levels of phospho-AKT in U87MG (compare lanes 1 and 2 of Fig. 1A) and U373MG (compare lanes 2 and 5 of Fig. 2C) cells are abrogated following the introduction of WT PTEN, whereas the expression of R130M and G129E mutants of PTEN in U87MG cells had no effect on the phosphorylation status of AKT (Fig. 1A, lanes 3 and 4). Lane 9 of the inset of Fig. 2C and lane 2 of Fig. 1A show the levels of WT PTEN in U373MG and U87MG cells, respectively.


Figure 1
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  		Figure 1. PTEN controls vitronectin-stimulated migration through protein phosphatase function. A, Western blots showing stable expression of WT PTEN (third panel from top), its mutants (G129E and R130M; third panel from top), and CSK (fourth panel from top), and effects of expression of these proteins on the levels of phospho-AKT (P473 AKT; top) in U87MG cells. Equal loading is confirmed by the levels of AKT (second panel from top) and β-actin (fifth panel from top). B, effect of WT PTEN, its mutants (G129E and R130M), and CSK on vitronectin-directed migration of U87MG glioma cells. Haptotaxis assays using different cell lines on 10 µg/mL vitronectin for 4 h show significant reduction of migration in U87MG cells expressing PTEN WT, G129E, and CSK, suggesting that the control of PTEN over migration is dependent on its protein phosphatase function and is mediated through SFKs. Columns, mean from five individual experiments; bars, SD.

 

Figure 2
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  		Figure 2. Vitronectin-stimulated migration in glioma cells (U87MG and U373MG) is abrogated by pharmacologic and physiologic inhibitors of SFKs and not by PI3K inhibition. A, effects of expression of PTEN and CSK and treatment with PP1 and LY294002 on levels of phospho-AKT (Ser473) in U87MG cells following vitronectin stimulation. Parental U87MG cells were treated with an inhibitor of SFK, PP1 (10 µmol/L; lane 4); or of PI3K, LY294002 (10 µmol/L; lane 3); versus no treatment (lanes 1 and 2). Stable clones of U87MG glioma cells expressing WT PTEN (lane 5) or CSK (lane 6) were also stimulated with vitronectin (10 µg/mL) for 30 min. Lane 1, no stimulation; lane 2, vitronectin stimulation. Equal loading is confirmed by the levels of AKT (second panel). Data show that PI3K inhibitor, LY294002, and PTEN overexpression significantly blocked AKT activation, whereas similar to PP1 treatment, CSK had no effect on AKT activation following integrin engagement (lanes 4 and 6). B, haptotaxis assays show virtually no effect of LY294002 on migration for 4 h at 10 µmol/L concentration, the concentration at which it causes significant reduction in activation of AKT (phospho-AKT immunoblot, lane 3, A). Conversely, PP1 dose dependently (1, 5, and 25 µmol/L) blocked migration of U87MG cells (PTEN null; B) without affecting phospho-AKT levels at 10 µmol/L concentration (A, lane 4). Columns, mean from five representative experiments; bars, SD. Individual levels of significance (P values) are shown. Data show that integrin ({alpha}vβ3/{alpha}vβ5)–directed migration in U87MG cells is independent of PI3K activity but dependent on SFK function. C, effects of expression of PTEN and treatment with PP1 and LY294002 on levels of phospho-AKT (Ser473) in U373MG cells following vitronectin stimulation. Parental U373MG (PTEN null) cells were treated (for 30 and 240 min) with an inhibitor of SFK, PP1 (10 µmol/L; lanes 4 and 7, respectively); or an inhibitor of PI3K, LY294002 (10 µmol/L; lanes 3 and 6, respectively); versus no treatment (lanes 1 and 2). Stable clones of U373MG glioma cells expressing WT PTEN (lane 5) were also stimulated with vitronectin (10 µg/mL) for 30 min. Lane 1, no stimulation; lane 2, vitronectin stimulation. Data show that LY294002 and PTEN overexpression significantly blocked AKT activation (lanes 3, 6, and 5, respectively), whereas PP1 treatment had no significant effect on AKT activation following integrin engagement (lanes 4 and 7). Equal loading of proteins is confirmed by the levels of AKT (second panel from top) and β-actin (third panel from top). Expression of WT PTEN in U373MG cells is confirmed by Western blot analysis (lane 9, inset). β-Actin was used as the loading control. D, haptotaxis assays using U373MG cells show no effect of LY294002 on vitronectin-directed migration (for 4 h at 10 µmol/L), the concentration and time at which it completely abrogated the activation of AKT (as shown in the phospho-AKT immunoblot, C, lane 6). Conversely, PP1 dose dependently (1, 5, and 25 µmol/L) blocked migration of U373MG cells (PTEN null). Columns, mean from five representative experiments; bars, SD. Individual levels of significance (P values) are shown. These data suggest that integrin ({alpha}vβ3/{alpha}vβ5)–directed migration in U373MG cells is independent of PI3K activity but is dependent on SFKs.

 
To investigate the effect of PTEN on integrin-directed migration, we conducted haptotaxis assays in stably transduced (a) U87MG cells (containing various mutants of PTEN) and (b) U373MG cells (containing WT PTEN). Haptotaxis was carried out under serum-free conditions to rule out the possibility of a combined "integrin-growth factor receptor–induced migration response" (e.g., the presence of platelet-derived growth factor, epidermal growth factor, or FBS would costimulate integrin and receptor protein tyrosine kinases to drive migration of glioma cells; ref. 24). As shown in Figs. 1B and 2D, vitronectin-directed migration is significantly reduced following the transduction of WT PTEN in both U87MG and U373MG cells, respectively. Interestingly, this abrogation of migration occurs to an equal extent in U87MG cells expressing G129E mutant of PTEN compared with cells expressing fully active WT PTEN. The fact that lipid phosphatase dead (but protein phosphatase active) mutant of PTEN (G129E) can inhibit vitronectin-directed migration in U87MG cells to the same degree as that of WT PTEN strongly suggests a role of PTEN's protein phosphatase activity in glioma cell migration and thus seems to be independent of PTEN's lipid phosphatase function (Fig. 1B). The validity of these assays was tested by (a) using function-blocking monoclonal antibodies (29), (b) determining the adhesion and proliferation of these cells under same assay conditions (29), and (c) determining the levels of integrin expressions in the presence of WT and different mutants of PTEN in these cells. From these data, we conclude that integrin-dependent migration in glioma cells is regulated by PTEN's protein phosphatase activity independent of the lipid phosphatase function.

CSK overexpression controls vitronectin-directed migration of U87MG cells. An important question then arose; if PTEN regulates migration via its control over protein phosphorylation events within the glioma cells, then what are the potential effectors for this action of PTEN? The results of other investigations have shown that the SFKs are important regulators of integrin-directed migration (24). As SFKs have been previously implicated in the regulation of integrin-directed migration and tumor metastasis, we investigated the effect of PTEN on SFKs in the control of migration of U87MG cells. It is well known that the function of SFKs is tightly regulated by another nonreceptor protein tyrosine kinase, CSK (13, 40). Because CSK physiologically regulates SFKs, overexpression of CSK in cells has been previously used as an excellent tool to examine the role of SFKs in various phenotypes. In our laboratory, we have also overexpressed CSK to explore the role of specific members of SFKs in neuronal differentiation, integrin-directed migration, and activation of AKT following growth factor stimulation (35). Hence, in this study, we decided to test the involvement of SFKs as potential downstream effectors of PTEN's protein phosphatase activity (that regulates glioma migration) by studying their migration on vitronectin following the overexpressing CSK in U87MG cells. We hypothesized that if PTEN regulates integrin-directed migration in a lipid phosphatase–independent but protein phosphatase–dependent manner, and if this effect is mediated through the control of PTEN over the activity of SFKs, then physiologic inhibition of SFKs (following CSK overexpression) in U87MG glioma cells (PTEN null) will cause a similar degree of inhibition of this phenotype to that of WT PTEN as well as its protein phosphatase active mutant (G129E mutant of PTEN). Hence, CSK was overexpressed to test the involvement of SFKs as downstream effectors of integrin-directed migration in glioma cells. Figure 1A (lane 5) shows stable overexpression of CSK in U87MG cells (4- to 5-fold increase in the expression of CSK as measured by Western blot) compared with the parental cell line (Fig. 1A, lane 1). We show (Fig. 1B) that stable CSK overexpression in these cells reduces their migration on vitronectin to a similar extent as that of the migration in cells containing WT PTEN and G129E mutant of PTEN, thus implicating the role of SFKs in integrin-directed migration. Interestingly, overexpression of CSK does not affect AKT activation (Fig. 2A, lane 6), confirming that the phenotypic changes related to CSK expression are likely not related to the changes due to the effect of SRC on the PI3K cascade. Furthermore, the integrin-mediated migration of glioma cells did not change following the inhibition of PI3K whereas it was inhibited by the treatment of the SFK inhibitor PP1. Treatment of LY294002 (10 µmol/L) significantly blocked the activation of AKT, whereas a similar treatment failed to affect migration of U87MG and U373MG glioma cells. These results support our concept that integrin-directed migration of glioma cells occurs in a PI3K-independent but SFK-dependent manner (Fig. 2). To further substantiate our results, we also determined the time course of vitronectin-stimulated activation of AKT in the presence of LY294002 (10 µmol/L) in U373MG cells and compared it with the migration of these cells under similar experimental conditions. Our data showed that despite the abrogation of phospho-AKT levels following 4 hours of LY294002 treatment, migration of U373MG cells on vitronectin remained unaffected (Fig. 2C and D).

The lipid phosphatase function of PTEN is required to control the PIP3-dependent activation of AKT in glioma cells. To understand the role of catalytic activities of different mutants of PTEN (G129E and R130M) in the control of downstream signals, we compared the enzymatic activity (in vitro phosphatase activities) of these mutants to their effects on the levels of phospho-AKT in glioma cells. We purified different mutants of PTEN (R130M, G129E) as GST fusion proteins in Escherichia coli and determined their lipid (Fig. 3A ) and protein (Fig. 3B) phosphatase activities compared with the WT PTEN and catalytically dead C124S mutant of PTEN. The G129E and R130M mutants displayed 7% and 5% of WT lipid (PIP3)–phosphatase activities and 65% and 12% of WT protein phosphatase activities, respectively (Fig. 3). In vitro lipid phosphatase and protein phosphatase activities of G129E and R130M mutants of PTEN support our argument that by expressing of these mutants in glioma cells, we were able to identify a PIP3-independent but protein phosphatase–dependent function of PTEN.


Figure 3
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  		Figure 3. Lipid phosphatase and protein phosphatase activities of WT PTEN and its mutants. A, lipid phosphatase activity of PTEN. The enzymatic activity of a GST fusion of WT PTEN and different mutants (G129E, R130M, and C124S) produced in E. coli cells were assayed using the phospholipid [water-soluble D-myo-PtdIns(3,4,5) P3] as substrate in vitro. Released phosphate was assayed colorimetrically using the malachite green reagent (absorbance values at 650 nm). Columns, mean absorbance from three independent assays in each case; bars, SD. Results show that G129E and R130M mutants display 7% and 5% of WT lipid PIP3 phosphatase activity. A catalytically dead mutant of PTEN (in which the active site cysteine was converted to serine, C124S) was used as an internal negative control. B, protein phosphatase activity of PTEN. The enzymatic activity of a GST fusion of WT PTEN and different mutants (G129E, R130M, and C124S) produced in E. coli cells was assayed using the protein (custom made acidic peptide YEEEEEpYEEEEEY) as substrate in vitro. Released phosphate was assayed colorimetrically using the malachite green reagent (absorbance values at 650 nm). Columns, mean absorbance from three independent assays in each case; bars, SD. Results show that G129E and R130M mutants display 65% and 12% of WT protein phosphatase activity. A catalytically dead mutant of PTEN (in which the active site cysteine was converted to serine, C124S) was used as an internal negative control.

 
PTEN controls FYN activity through its protein phosphatase function. To explore the role of PTEN in the control of SFKs, we confirmed the presence of specific members of SFKs (FYN, LYN, SRC, and YES) in U87MG cells in agreement with previous report (24). To determine the specificity of PTEN's protein phosphatase activity in FYN dephosphorylation, we examined the effect of WT PTEN, R130M mutant of PTEN (catalytically dead PTEN), G129E mutant of PTEN (lipid phosphatase dead, protein phosphatase active PTEN), and CSK on integrin-dependent activation of FYN kinase in glioma cells. Immunoprecipitation of activated FYN following vitronectin stimulation of U87MG cells (PTEN null) shows robust activation of FYN kinase, an event abrogated by the reconstitution of WT and G129E mutant of PTEN. Integrin engagement caused 40% increase of (above basal level) FYN kinase activity that occurred at the same time (15 minutes) as that of the increase in FYN phosphorylation (Y418 FYN) following integrin stimulation as shown in Fig. 4A and B . This activation of FYN kinase is significantly reduced in WT PTEN–reconstituted glioma cells following similar integrin engagement (Fig. 4B), which is comparable with the decrease in FYN phosphorylation (Fig. 4A). Figure 4B shows that FYN kinase activity is also inhibited significantly in U87MG cell line expressing G129E mutant of PTEN. In sharp contrast, the overexpression of catalytically dead PTEN (R130M) has no effect on the activation of FYN kinase following integrin stimulation. As expected, overexpression of CSK (a physiologic inhibitor of SFK) blocked the kinase activity of FYN following integrin stimulation (Fig. 4B). CSK overexpression in U87MG cells inhibited FYN kinase activity downstream of {alpha}vβ3 engagement (comparable with the treatment of PP1; data not shown), thus suggesting the role of this member of SFKs in {alpha}v integrin–mediated glioma migration (Fig. 4B). Our results indicate that the effect of PTEN on {alpha}v integrin–directed migration is correlated with the effect of PTEN on {alpha}v integrin–directed FYN activation in glioma cells. FYN protein levels in U87MG cells reconstituted with (a) WT PTEN, (b) no PTEN, (c) G129E and R130M mutants of PTEN, and (d) CSK at different time points of integrin stimulation (15 and 30 minutes) remained unaltered as shown by Western blots performed on cell lysates (Fig. 4B). In addition, the effect of PTEN seems to be integrin specific, as its control over FYN is less pronounced with fibronectin stimulation, suggesting specificity of either SFK members or other pathways that are specific to {alpha}vβ3 or {alpha}5β1 activation (21, 41). Our data provide evidence for the fact that the FYN kinase activity (predominantly expressing member of SFKs) in glioma cells is reduced in a PTEN (specifically involving the protein phosphatase function PTEN)–dependent manner upon {alpha}v stimulation (which correlates to the integrin-dependent migration) and compares this effect to the phenotypic and biochemical effects of CSK overexpression (biological inhibitor of SFKs) and treatment of PP1 (pharmacologic inhibitor of SFKs). Together, our data indicate that the control of PTEN over integrin-directed migration in glioma cells is mediated through SFKs (specifically FYN) and its downstream effectors.


Figure 4
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  		Figure 4. PTEN controls integrin-induced activation of specific SFK, FYN, and RAC1 GTPase activity in glioma cells. A, immunoprecipitated FYN kinase following {alpha}vβ3 stimulation from PTEN null and WT PTEN reconstituted U87MG glioma cells at different time points (15 and 30 min). Western blot analyses show phospho-FYN (Y418, top) and total FYN (bottom) after vitronectin stimulation. The results provide evidence that PTEN abrogates FYN phosphorylation/activation following integrin stimulation in glioma cells. NS, no stimulation. FYN Western blot (bottom) shows an equal amount of FYN that was immunoprecipitated with antibody following vitronectin engagement. Equal loading is confirmed by the levels of total FYN (second panel from top). B, effects of PTEN, catalytically dead PTEN (R130M), lipid phosphatase dead PTEN (G129E), and CSK overexpression on FYN kinase activity following integrin stimulation (vitronectin engagement) in U87MG cells. FYN kinase activity was determined from 100 µg protein derived from U87MG cell lysates. Phosphotransferase activity toward FYN-specific peptide was determined under vitronectin stimulation. Data are expressed as cpm for kinase activity, represented by an immunoprecipitation performed in U87MG cells transduced with empty vector (U87MG null), WT PTEN, catalytically dead PTEN (R130M), lipid phosphatase dead PTEN (G129E), and CSK. NS, no stimulation; 15, 15-min stimulation on vitronectin; 30, 30-min stimulation on vitronectin-coated plates. Bottom, Western blot analysis for the expression of total FYN protein in the lysate from which each immunoprecipitation was performed. Columns, original cpm values from three individual experiments. C, to assess the downstream effects of PTEN's control of SFK, we show that PTEN regulates glioma cell motility and controls integrin-induced activation of the GTPase, RAC1. We measured the conversion of GDP-RAC1 to the activated GTP RAC1 in WT PTEN–reconstituted, R130M PTEN–reconstituted, G129E PTEN–reconstituted, and CSK-overexpressed U87MG glioma cells following stimulation with vitronectin at the indicated times. Levels of GTP-bound RAC were measured by performing pull-down experiments using GST fusion protein representing the GTP-RAC1 binding CRIB domain of PAK kinase. Equal loading is confirmed by the levels of total RAC1 (second panel from top). It is noted that under experimental conditions, when PTEN retains its protein phosphatase activity (as in WT and G129E mutant), it can exert its control over the activation of RAC1 as well as cell migration on vitronectin. In contrast, loss of PTEN's protein phosphatase activity (as in null and catalytically dead mutant of PTEN, R130M) is associated with the failure of PTEN to control migration, FYN kinase activity, and activation of RAC1. From these data, we suggest that PTEN's protein phosphatase activity is necessary to control integrin-directed migration through FYN kinase and RAC1 GTPase in glioma cells.

 
The protein phosphatase function of PTEN controls migration and the activation of RAC1. We argue that if PTEN controls glioma migration in a lipid phosphatase–independent manner via SFKs, then this event will involve downstream effectors that mediate both SFK activation and integrin-directed migration. RAC1 is one of the well-established downstream effectors of integrin-mediated migration and SFKs activation in various cells (23, 24). Here, in this study, we show that in glioma cells, activation of RAC1 following vitronectin stimulation is controlled by PTEN (Fig. 4C). Reconstitution of WT PTEN in U87MG cells almost completely abrogates levels of RAC1-GTPase following integrin engagement, and more importantly, this effect, like the control of vitronectin-mediated migration by PTEN, occurs in a lipid phosphatase–independent manner. Reconstitution of U87MG cells with protein phosphatase active and lipid phosphatase dead (G129E) mutant of PTEN caused a comparable inhibition of RAC1 activation following integrin engagement. We and others have established the role of SFKs in RAC1 signaling (35). Based on the above model for SFK action, we studied the effect of CSK overexpression on {alpha}vβ3 integrin–directed RAC1 activation in glioma cells. Levels of activated RAC1 are almost completely abrogated following CSK overexpression under integrin stimulation, suggesting that the activation of RAC1 in glioma migration is a downstream event of integrin-mediated activation of SFKs. Further, abrogation of migration in glioma cells following overexpression of CSK (Fig. 1B) was comparable with the inhibition of the RAC1 activity under similar conditions of integrin stimulation (Fig. 4C). These results indicate that the protein phosphatase activity of PTEN controls integrin-directed migration in glioma cells by regulating activation of RAC1 downstream of activation of SFKs.

Effect of Pten+/- status on vitronectin-directed migration of primary astrocytes. To validate our observations regarding the effect of PTEN on the migration of glioma cells, we sought to determine the effect of genetic loss of PTEN on the integrin-directed migration using Pten haploinsufficient (Pten heterozygous mice) primary murine astrocytes. Figure 5A shows levels of phospho-AKT in WT versus Pten heterozygous astrocytes. In wound healing (scratch assay) assay, cultured astrocytes were allowed to migrate on vitronectin for 24 hours (Fig. 5B). Astrocytes that are wild-type for PTEN migrate into the wound more slowly than Pten heterozygous astrocytes, confirming the biological effects of Pten haploinsufficiency in the control of integrin-directed migration. To determine the contribution of lipid phosphatase activity of PTEN in the control of astrocyte migration, we stably overexpressed AKT in primary WT astrocytes. We argue that if activation of AKT is important in glioma migration, then astrocytes with stable high levels of phospho-AKT would display greater motility than WT astrocytes. On the contrary, our results show that even high levels of phospho-AKT in WT primary astrocytes have nominal effect on {alpha}v integrin–directed migration as shown in Fig. 5A and B. In summary, these results further support our working model that the regulation of integrin-directed glioma migration is independent of PTEN's lipid phosphatase activity and AKT kinase activity.


Figure 5
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  		Figure 5. PTEN regulates vitronectin-mediated cell movement in an AKT-independent manner in primary astrocytes and FYN is involved in the migration of U87MG cells. A, PTEN inhibits {alpha}vβ3/{alpha}vβ5–directed migration in primary astrocytes; the overexpression of WT AKT does not antagonize the effect of PTEN on migration. Primary astrocytes were isolated from WT mice or mice heterozygous for Pten as described in Materials and Methods. WT AKT (WT-aAKT) was overexpressed in WT astrocytes to determine the role of the PI3K pathway in migration. Immunoblots reveal that the level of phosphorylated AKT is lower in PTEN WT astrocytes (lane 2) than in the PTEN heterozygous astrocytes (lane 1). The level of phosphorylated AKT is significantly higher in astrocytes that overexpressed WT AKT (lane 3) compared with WT astrocytes (lane 2). Equal loading is confirmed by the levels of AKT (second panel from top) and β-actin (third panel from top). B, WT astrocytes with and without overexpressing WT AKT and astrocytes from Pten heterozygous mice were stained with crystal violet following vitronectin-stimulated scratch assay for 24 h. WT AKT overexpression in astrocytes had no significant effect on migration, whereas Pten heterozygous astrocytes migrated significantly faster than WT astrocytes. Results were quantified for statistical significance. Columns, mean; bars, SD. Data are representative of four individual experiments. *, P < 0.01. Results indicate that the control of PTEN over vitronectin-mediated cell movement is independent of the state of activation of AKT. C, effect of FYN on integrin-directed migration of U87MG cells. FYN was knocked down in U87MG cells using siRNA. Cells were transfected with FYN-specific siRNA (SMART pool FYN) or control siRNA (nonspecific control pool) for 24 and 48 h. Cell lysates were used to test the change in the levels of expression of FYN protein by Western blot (top). β-Actin was used as loading control (bottom). D, the effect of FYN on integrin-directed migration in U87MG glioma cells was determined by studying the migration of U87MG cells on vitronectin following the transfection of FYN-specific siRNA. After 24 and 48 h of transfection with FYN-specific siRNA, the cells were subjected to the vitronectin-directed migration. Columns, mean; bars, SD. Data are representative of three individual experiments. *, P < 0.05. Results show that at both time points (24 and 48 h) following transfection with FYN-specific siRNA, migration of U87MG cells was significantly blocked.

 
FYN regulates vitronectin-directed migration in both glioma cells and primary astrocytes. To determine the role of FYN kinase in {alpha}v integrin–directed migration, we studied migration of both glioma cells and primary astrocytes using siRNA and genetic approaches, respectively. FYN kinase was knocked down in U87MG cells using siRNA (Fig. 5C). Migration of these cells was found to be significantly blocked compared with the respective control (Fig. 5D). To further substantiate our observation, we also compared the migration of primary astrocytes (fourth passage) from Fyn–/– mice to the migration of WT astrocytes (similar passage). Figure 6A shows that {alpha}vβ3 integrin–dependent migration of astrocytes derived from Fyn–/– mice was significantly reduced (60%) compared with the migration of WT astrocytes. To obtain evidence that FYN kinase is involved in PTEN-mediated inhibition of integrin-directed migration of glioma cells, we studied the migration (on vitronectin) of primary astrocytes from (a) Pten heterozygous, (b) Fyn heterozygous, and (c) Pten and Fyn double heterozygous mice. Figure 6B shows that the migration of astrocytes from Pten heterozygous mice was significantly higher than their WT counterpart, whereas Fig. 6C shows that the migration defect observed in Fyn haploinsufficient astrocytes on {alpha}vβ3/{alpha}vβ5 was reversed in mice haploinsufficient for Pten (Pten and Fyn double heterozygous condition). These combined results, including the data obtained from the genetically altered mice, strongly suggest that the inhibitory effect of PTEN on integrin-directed migration of glioma cells is mediated through its protein phosphatase function and involves FYN kinase.


Figure 6
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  		Figure 6. FYN and PTEN control vitronectin-directed migration in primary astrocytes. A, FYN controls {alpha}vβ3/{alpha}vβ5–directed migration in primary astrocytes. Vitronectin-directed haptotaxis assay (24 h) was carried out in neonatal astrocytes (passage 4) from Fyn–/– [knockout (KO)] and Fyn+/+ (WT) mice. Representative photomicrographs of vitronectin-directed migration and its semiquantification show that migration of the neonatal astrocytes derived from Fyn knockout mice was significantly blocked compared with WT astrocytes. Columns, mean from five individual experiments; bars, SD. *, P < 0.003. B, PTEN controls {alpha}vβ3/{alpha}vβ5–directed migration in primary astrocytes. Vitronectin-directed haptotaxis assay (24 h) was carried out in neonatal astrocytes (fourth passage) from Pten+/– (heterozygous) and Pten+/+ (WT) mice. Results show that the astrocytes derived from Pten heterozygous mice migrated significantly faster compared with the WT astrocytes. Columns, mean from five individual experiments; bars, SD. *, P < 0.01. C, the inhibitory effect of genetic loss of Fyn (Fyn heterozygous condition) on {alpha}vβ3/{alpha}vβ5–directed migration in primary astrocytes is blocked by the haploinsufficiency of Pten (Pten and Fyn double heterozygous condition). Vitronectin-directed haptotaxis assay (24 h) was carried out in neonatal astrocytes (fourth passage) from Fyn+/– (heterozygous; FYN Het), Pten and Fyn double heterozygous (FYN-Het PTEN-Het), and WT mice. Results show that the astrocytes derived from Fyn heterozygous mice migrated significantly slower compared with the WT astrocytes, whereas the astrocytes derived from Pten and Fyn double heterozygous mice migrated significantly faster compared with the Fyn heterozygous astrocytes. Columns, mean from five individual experiments; bars, SD. Results show that the genetic loss of Fyn inhibits the vitronectin-directed migration, whereas the haploinsufficiency of Pten counteracts this effect. Together, these results suggest that PTEN regulates vitronectin-directed migration via its control on FYN kinase.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Malignant gliomas are one of the most frequently occurring primary brain tumors in adults and in their most advanced stage (GBM) are uniformly fatal in both adults and pediatric patients (42, 43). Histologic hallmarks of advanced-stage gliomas give clues to the oncogenic cellular milieu that include increased vascularization (angiogenesis), high mitotic index (proliferation), and insidious cell dispersal through normal brain parenchyma (migration and invasion; ref. 44). Understanding signal transduction pathways involving cell motility as it relates to the increased migration and invasion in GBMs is critical to mapping out potential new therapies for these aggressive tumors.

Interactions between SRC and PTEN have recently been documented in breast cancer and shown to have significant implications not only in the malignant phenotype but also in treatment responses (32–34). The ability of SRC to phosphorylate PTEN is correlated with decreased membrane localization, reduced activity, and increased growth, promoting the downstream effect of activating the PI3K pathway (32, 33). The use of tumor-associated somatic mutations in PTEN to evaluate its biochemical functions has been a useful tool to dissect the lipid versus protein phosphatase functions of PTEN in mammalian cells. Only a few investigations have used cell lines that were stably transduced with PTEN or mutants of PTEN (5, 10, 45) to address the role of PTEN in tumor progression. We established a relationship between PTEN (protein phosphatase–active PTEN) and SFKs (FYN) in the control of integrin-directed haptotaxis of malignant gliomas. Our data show that, in malignant gliomas, the relationship between PTEN and SFKs may be reciprocal. The results of our study suggest that the control of PTEN over FYN kinase activity functionally correlated with the inhibition of the migration of these cells in a well-established in vitro migration system (29, 30). Further support that PTEN's protein phosphatase function regulates vitronectin-stimulated migration in glioma is obtained from the result that PP1 treatment and CSK overexpression inhibited the migration without affecting levels of phospho-AKT in these cells. In reports of Gu et al. (13), CSK expression has been shown to have little effect on the migration of U87MG cells, despite increased phosphorylation of SRC and its resulting inactivated form; however, these studies implemented transient transfection, with 7.5-fold overexpression, and fibronectin stimulation. Stable expression of CSK may more closely resemble physiologic conditions, allowing CSK to tightly regulate SFKs upon activation. RAC GTPases are known to promote motility and have previously been shown to be involved in integrin-mediated migration through interactions with FAK, MAPK, and SRC (35, 46). Our findings show that in addition to controlling SFKs, PTEN also controls RAC1 GTPase activity in a lipid phosphatase–independent manner. Activation of AKT has long been viewed as the primary effector of an up-regulated PI3K pathway, and as such, is an excellent surrogate marker for PTEN's lipid phosphatase function (5). We have observed that the overexpression of AKT alone transforms primary murine astrocytes5 but has no effect on the migration of these cells on vitronectin (Fig. 5B). Because PTEN controls AKT, these data provide additional evidence that PTEN's lipid phosphatase activity does not control glioma cell motility.

The ability of PTEN to control migration is more pronounced through vitronectin, which binds to {alpha}vβ3 integrin, than through collagen. Hence, it is clear that specific integrins provide signaling cues that ultimately mediate specific phenotypic changes. The use of stable transfection of PTEN or PTEN mutants into U87MG cells and U373MG cells has allowed us to determine that PTEN's protein phosphatase activity seems to control migration and coordinately regulate SFKs and RAC1 GTPases. It is not known whether PTEN controls migration through autodephosphorylation or through dephosphorylation of another protein substrate (1, 47). Recently, we have isolated novel PTEN's mutants by alanine scan methods that only affect PTEN, protein phosphatase activity (but are still able to dephosphorylate PIP3). These will be useful in providing direct evidence of PTEN's protein phosphatase activity, as well as its specific substrates in biological systems.

Previous studies have implicated PTEN's lipid phosphatase activity and its control of the PI3K pathway in controlling migration of certain cell types (48, 49). For instance, in mouse embryonic fibroblasts (MEF), Liliental et al. used retroviral transfection of WT, catalytically dead (C124S) and lipid dead (G129E) mutants into PTEN-null MEFs to evaluate cell motility using a wound healing assay on fibronectin. Their approach differs from ours in that they studied MEFs and a different matrix protein-integrin interaction via {alpha}5β1. We and others have observed cell type–specific differences in the effect of PTEN on haptotactic migration, in that certain primary cell types (macrophages and endothelial cells) use PI3K as a requirement for integrin-dependent cell movement.5 In addition, some discordant results have been seen regarding the dual phosphatase effect of PTEN on growth and cell cycle regulation in different cell types. Nevertheless, evidence for the importance of PTEN's protein phosphatase activity in mammalian physiology is mounting (21, 38, 47, 49, 50). In this study, we provide evidence that PTEN's protein phosphatase function is necessary and sufficient to regulate integrin-directed migration of glioma cells. The detailed mechanism of involvement of the downstream signals initiated by the protein phosphatase action of PTEN for this phenotype in glioma cells remains an open question. Although results of our study suggest an involvement of downstream effectors like SFKs and RAC1 in this signaling pathway, the data are correlative and does not necessarily prove that the regulatory effect of PTEN on the migration of glioma cells is mediated specifically and solely via its control on FYN kinase. In summary, our data showing that the blockade of (a) kinase activity of FYN, (b) activation of RAC1, and (c) vitronectin-directed migration in glioma cells following the overexpression of CSK, together with the results of studies using FYN siRNA, strongly indicate that FYN kinase is a downstream link between the protein phosphatase activity of PTEN and glioma migration. Here, we have identified a novel AKT-independent pathway linking protein phosphatase activity of PTEN to vitronectin-directed migration in glioma cells. One relevant issue regarding the involvement of FYN kinase in PTEN-mediated (protein phosphatase activity of PTEN), vitronectin-dependent migration of glioma is to identify the downstream effectors of FYN. Interestingly, our initial data provide correlative evidence to suggest the role of RAC1 in this signaling pathway. Currently, studies are ongoing in our laboratory to evaluate the specific role of RAC1 in this novel AKT-independent pathway linking protein phosphatase activity of PTEN to the integrin-dependent migration in gliomas. Further studies are required to delineate the entire pathway in a systematic fashion by manipulating each and every component of the pathway.

In conclusion, our findings implicate PTEN in mediating important functions of malignant tumor cells via catalytic activities other than its ability to dephosphorylate PIP3. PTEN loss is observed in many malignant tumors and is a marker for advanced neoplastic disease, associated with a highly aggressive, resistant, and frequently invasive or metastatic cancer. Targeted therapies are being evaluated in patient populations who are segregated based on PTEN status and the expected effect of the treatment on the PI3K/AKT pathway. PI3K inhibitors are currently under development in attempt to pharmacologically mimic PTEN tumor suppressor activity. However, if PTEN's protein phosphatase activity plays a role in controlling the motile function of cells, then inhibitors targeting the downstream effectors of PI3K and the AKT pathway may not be as effective as expected. Future deliberation on the ability of PTEN to regulate SFKs and RAC-GTPases will be important in identifying other targets associated with complete loss of PTEN's tumor suppressor functions. From our results, we would predict that PTEN loss in glioblastoma will be associated with up-regulation of not only PI3K- but also SFK-mediated pathways that are involved in migration and invasion. A potential clinical application would therefore be to combine a PI3K inhibitor (such as SF1126; ref. 51) with an SFK inhibitor (e.g., dasatanib) to increase the efficacy of these targeted therapies in this and other malignant motile diseases.


   Acknowledgments
 
Grant support: NIH grant CA94233 (D.L. Durden), Georgia Cancer Coalition (D.L. Durden), Golfers Against Cancer, Goldhirsh Foundation, Cure Childhood Cancer Foundation, and Aflac Cancer Center and Blood Disorder Services.

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 Dr. Yue Feng (Emory University) for the Fyn–/– mice and Bernadette Jean-Joseph for technical support.


   Footnotes
 
5 Unpublished observation. Back

Received 3/28/07. Revised 11/30/07. Accepted 12/31/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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The Mechanical Rigidity of the Extracellular Matrix Regulates the Structure, Motility, and Proliferation of Glioma Cells
Cancer Res., May 15, 2009; 69(10): 4167 - 4174.
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Y. Zhu, A. Wloch, Q. Wu, C. Peters, A. Pagenstecher, H. Bertalanffy, and U. Sure
Involvement of PTEN Promoter Methylation in Cerebral Cavernous Malformations
Stroke, March 1, 2009; 40(3): 820 - 826.
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