Ionizing Radiation Enhances Matrix Metalloproteinase-2 Secretion

and Invasion of Glioma Cells through Src/Epidermal Growth

Factor Receptor–Mediated p38/Akt and Phosphatidylinositol

3-Kinase/Akt Signaling Pathways

Chang-Min Park,1Myung-Jin Park,

1Hee-Jin Kwak,

1Hyung-Chahn Lee,

1Mi-Suk Kim,

4

Seung-Hoon Lee,4In-Chul Park,

1Chang Hun Rhee,

1,2and Seok-Il Hong

1,3

1Laboratory of Functional Genomics, 2Department of Neurosurgery, and 3Laboratory Medicine and Clinical Pathology, Korea Institute ofRadiological and Medical Sciences, Seoul, Korea and 4Research Institute and Hospital, National Cancer Center, Goyang, Gyeonggi, Korea

Abstract

Glioblastoma is a severe type of primary brain tumor, and itshighly invasive character is considered to be a major ther-apeutic obstacle. Several recent studies have reported thationizing radiation (IR) enhances the invasion of tumor cells,but the mechanisms for this effect are not well understood. Inthis study, we investigated the possible signaling mechanismsinvolved in IR-induced invasion of glioma cells. IR increasedthe matrix metalloproteinase (MMP)-2 promoter activity,mRNA transcription, and protein secretion along with theinvasiveness of glioma cells lacking functional PTEN (U87,U251, U373, and C6) but not those harboring wild-type (WT)-PTEN (LN18 and LN428). IR activated phosphatidylinositol3-kinase (PI3K), Akt, and mammalian target of rapamycin,and blockade of these kinases by specific inhibitors (LY294002,Akt inhibitor IV, and rapamycin, respectively) and transfectionof dominant-negative (DN) mutants (DN-p85 and DN-Akt) orWT-PTEN suppressed the IR-induced MMP-2 secretion in U251and U373 cells. In addition, inhibitors of epidermal growthfactor receptor (EGFR; AG490 and AG1478), Src (PP2), and p38(SB203580), EGFR neutralizing antibody, and transfection ofDN-Src and DN-p38 significantly blocked IR-induced Aktphosphorylation and MMP-2 secretion. IR-induced activationof EGFR was suppressed by PP2, whereas LY294002 andSB203580 did not affect the activations of p38 and PI3K,respectively. Finally, these kinase inhibitors significantlyreduced the IR-induced invasiveness of these cells on Matrigel.Taken together, our findings suggest that IR induces Src-dependent EGFR activation, which triggers the p38/Akt andPI3K/Akt signaling pathways, leading to increased MMP-2expression and heightened invasiveness of PTEN mutantglioma cells. (Cancer Res 2006; 66(17): 8511-9)

Introduction

Malignant glioma, the most common primary central nervoussystem tumor, is one of the most aggressive and lethal neoplasms.One of the major limitations in the treatment of glioma is the highprevalence of intrinsic or acquired resistance against radiation (1),

which is likely associated with intrinsic cellular radioresistance,rapid cellular proliferation, and high invasiveness (2, 3). Amongthese biological factors, local invasive growth is a key feature ofmalignant brain tumors, showing a high incidence of recurrenceeven following drastic surgical resection (4, 5). Thus, it is importantto understand the mechanisms of tumor invasiveness within brain,because this may lead to new strategies for curative treatment ofglioma.Tumor cell invasion involves the proteolytic degradation of

extracellular matrix (ECM) components by tumor cell-secretedproteases, including serine proteases, plasminogen activators, andmatrix metalloproteinases (MMPs; refs. 6, 7). Elevated levels ofMMPs have been found in many tumors and are believed to play animportant role in cellular invasion and metastasis (6–8). Previousstudies have shown that glioma cells secrete MMPs and theirendogenous inhibitors, tissue inhibitors of metalloproteinases(TIMPs), and that high MMP expression is correlated with highaggressiveness in brain tumors (7, 9–11). In particular, increasedexpression and activity of MMP-2 (72-kDa gelatinase A) andMMP-9 (92-kDa gelatinase B) have been correlated with anincreased grade of glioma malignancy (10, 12–16). Expression ofMMP-2 is high in glioma cells in vitro and seems to be correlatedwith the invasive character of these cells (12–14), whereas MMP-9expression is induced by various stimuli, such as inflammatorycytokines and growth factors (15, 16). Thus, MMP-2 and MMP-9 areintriguing candidates for therapeutic manipulation in invasivegliomas.Glioma invasiveness may be mediated by some of the genetic

events commonly found in these tumors. In gliomas, tumorsuppressor genes, such as PTEN and p53 , are frequently mutated ordeleted, whereas oncogenes, such as epidermal growth factorreceptor (EGFR) and platelet-derived growth factor receptor(PDGFR), are commonly overexpressed. These genetic alterationscan lead to activation of invasiveness-related signal transductionpathways, including focal adhesion kinase (FAK), Ras/mitogenactivated protein kinase (MAPK), and phosphatidylinositol3-kinase (PI3K)/Akt pathways (17, 18). The latter pathway isregulated by PTEN (also called MMAC1), a tumor suppressor genelocated on human chromosome 10q23.3 (19), which is known toregulate cell growth, apoptosis (20), and interactions with the ECM,and has been shown to inhibit cell migration, spreading, and focaladhesion (21). PTEN dephosphorylates two lipid signal transduc-tion molecules (phosphatidylinositol 3,4,5-triphosphate and phos-phatidylinositol 3,4-bisphosphate) involved in the PI3K/Aktpathway (22) and exhibits protein phosphatase with dual specificityin vitro against its potential cellular target, FAK (23). PTEN has

Note: C-M. Park and M-J. Park contributed equally to this study.Requests for reprints: Seok-Il Hong, Laboratory of Functional Genomics,

Laboratory Medicine and Clinical Pathology, Korea Institute of Radiological andMedical Sciences, 215-4, Gongneung-Dong, Nowon-Ku, Seoul 139-706, Korea. Phone:82-2-970-1260; Fax: 82-2-978-2005; E-mail: hongsicp@kcch.re.kr.

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-4340

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been shown to suppress MMP-2 expression and invasion of gliomacells in a phosphatase-dependent manner (24, 25) and has recentlybeen reported to suppress mutant EGFR type III–mediatedinvasion of glioma cells by blocking FAK phosphorylation (26).Furthermore, we recently showed that hyaluronic acid inducesexpression of MMP-9 and osteopontin and consequent invasionand/or migration of glioma cells and that these events required theprotein and lipid phosphatase activities of PTEN, respectively(25, 27). Collectively, these earlier findings suggest that the PI3K/Akt pathway might be a good target for new strategies aimed atcontrolling glioma invasion and migration.Various stimuli, such as ionizing radiation (IR), growth factor/

receptor interactions, and ECM/integrin interactions, have beenshown to increase the invasiveness of glioma cells (28–31).Exposure of cancer cells to IR stimulates the expression of MMP-2,MMP-9, Bcl-2, and integrin family members and activates multiplesignaling pathways involved in controlling cell survival and repo-pulation (28–34). However, the precise regulatory mechanismsresponsible for IR-induced MMP expression and cancer cellinvasion are not well understood.In the present study, we investigated the some possible

mechanisms for the effect of IR on MMP-2 secretion and invasionof glioma cells and found that IR up-regulates glioma cell invasionby inducing MMP-2 and that the Src/EGFR-mediated p38/Akt andPI3K/Akt pathways are critically involved in these events. Ourfindings suggest that these signaling pathways could be potentialtargets for improving the efficacy of radiotherapy for malignantgliomas.

Materials and Methods

Reagents and cell culture. Antibodies against EGFR, phosphorylatedEGFR, PTEN, Src, and hemagglutinin (HA) were purchased from Santa

Cruz Biotechnology (Santa Cruz, CA). Antibodies against MMP-2, MMP-9,TIMP-1, and TIMP-2 were from Calbiochem (La Jolla, CA). Antibodies

against Akt, p38, and mammalian target of rapamycin (mTOR), and

phosphorylated form of Src, Akt, mTOR, and p38 were from Cell Signaling

Biotechnology (Beverly, MA). Antibody against p85 subunit of PI3K waspurchased from Upstate Biotechnology (Lake Placid, NY). Specific

inhibitors of EGFR (AG490 and AG1478), ErbB2 (AG825), PDGFR

(SU5402), PI3K (LY294002), Src (PP2), mTOR (rapamycin), MAPK family[PD98059, SB203580 and SP600125, extracellular signal-regulated kinase 1/2

(ERK1/2), p38 and c-Jun NH2-terminal kinase (JNK), respectively], Akt

(Akt inhibitor IV), MMP-2 (OA-Hy), and a broad-spectrum kinase inhibitor

(genistein) were purchased from Calbiochem. Human glioma cell lines[U87, U251, and U373 (obtained from American Type Culture Collection)

and LN18 and LN428 (generously gifts from Dr. Funari, Ludwig Institute for

Cancer Research, La Jolla, CA)] and rat glioma cell line C6 were maintained

in DMEM supplemented with 10% heat-inactivated fetal bovine serum,100 units/mL penicillin, and 100 Ag/mL streptomycin at 37jC in a humi-

dified atmosphere containing 5% CO2.

Irradiation. Cells were plated in 30-mm dishes and incubated at 37jCunder humidified 5% CO2 and 95% air in culture medium until 70% to 80%

confluent. Cells were then exposed to g-rays from a 137Cs g-ray source[Atomic Energy of Canada, Korea Institute of Radiological and Medical

Sciences (KIRAMS), Seoul, Korea] at a dose rate of 3.81 Gy/min.Transfection and transduction. Wild-type (WT)-PTEN (obtained from

Dr. Sun, Yale University, New Haven, CT), mutant PTEN (C124S, protein and

lipid phosphatase activity-deficient mutant; generously gift from Dr. Whang,

University of North Carolina School of Medicine, North Carolina, NC),dominant-negative (DN) mutant of p85 [DN-p85 (regulatory domain of

PI3K)], DN-Src, DN-Akt (generous gift from Dr. Su-Jae Lee, KIRAMS, Seoul,

Korea), and WT-MKK6 (generous gift from Dr. Aree Moon, Duksung

Women’s University, Seoul, Korea) plasmids were transiently transfected

into U251 and U373 glioma cells using Effectene transfection reagent(Qiagen, Valencia, CA) by following the supplier’s instructions. For

retrovirus transduction, U251 and U373 cells were infected with control

retrovirus and retrovirus harboring DN-38 and WT-p38 (generous gift from

Dr. Su-Jae Lee) for 90 minutes at 37jC. In adenoviral transduction, cellswere infected with 50 plaque-forming units per cell of control adenoviral

LacZ and adenoviral WT-EGFR (generous gift from Dr. Seung-Hoon Lee,

National Cancer Center, Goyang, Gyeonggi, Korea) for 90 minutes at 37jC.After 48 hours, transfected and transduced cells were used for furtherexperiments. Expression of target genes was confirmed by Western blot

analysis using the antibodies against target proteins, phosphorylated

downstream effector molecules, and/or HA.

Reverse transcription-PCR (RT-PCR). Total RNA was isolated fromglioma cells irradiated together with or without actinomycin D (a tran-

scription blocker) or cyclohexamide (a translation blocker) using the

RNeasy kit (Qiagen), and complimentary first-strand DNA was generatedusing the reverse transcription system kit (Bioneer, Daejeon, Korea)

according to the manufacturer’s protocol. The PCR conditions involved

an initial denaturation step at 94jC for 5 minutes followed by 30 cycles at94jC for 30 seconds, 55jC for 30 seconds, and 72jC for 30 seconds.Oligonucleotide primer sequences used were as follows: MMP-2 sense

5 ¶-CAGGCTCTTCTCCTTTCACAAC-3¶ and antisense 5¶-AAGCCACGG-CTTGGTTTTCCTC-3¶ and h-actin sense 5¶-CATGGGTCAGAAGGATTCCTAT-3¶and antisense 5¶-GCGCTCGGTGAGGATCTTCAT-3¶. PCR was done using the PCRMaster kit (Bioneer) according to the manufacturer’s protocol.

Luciferase reporter assays. pGL3 luciferase reporter vector, containingMMP-2 promoter regions, was provided by Dr. Sang-Oh Yoon (KoreaAdvanced Institute of Science and Technology, Taejon, Korea). pRL-

cytomegalovirus (CMV) vector (Promega, Madison, WI) was used to

evaluate transfection efficiency. Cells were seeded in 12-well plates and

cultured at 37jC until they reach 50% to 60% confluency. Plasmids werethen transiently cotransfected into glioma cells by using Effectene (Qiagen).

After 24 hours, cells were irradiated at 5 Gy, and cell lysates were prepared

at 6, 12, and 24 hours of incubation using Dual-Luciferase Assay kit

(Promega). The luminescence was measured by using MicroLumatPlusLB96V microplate luminometer (EG&G Berthold, Wellesley, MA) according

to the manufacturer’s protocol.

Gelatin zymography. Glioma cells in subconfluent culture (f70-80%

cell density of confluent culture) were washed and refreshed with serum-

free DMEM and then irradiated and incubated for 18 hours. In some experi-

ments, cells were preincubated for 1 hour with various kinase inhibitors

before irradiation. The enzymatic activity of electrophoretically separated

gelatinolytic enzymes in the conditioned medium of glioma cells were

determined by gelatin zymography as described previously (25). Zones of

gelatinolytic activity were detected as clear bands against a blue background.

Densitometric analysis was done using Scion Image NIH Image program.

Western blot analysis. Glioma cells were irradiated at various dose andlysed in lysis buffer as described previously (25). After a brief sonication, the

lysates were clarified by centrifugation at 12,000 � g for 15 minutes at 4jC,and protein content was measured by the Bradford method. An aliquot

(50 Ag protein/lane) of the total protein was separated by 10% or 12% SDS-

PAGE and blotted to nitrocellulose membrane (0.2 Am; Amersham,Arlington Heights, IL), and Western blot was done as described previously(25). Immunoblots were visualized by enhanced chemiluminescence

(Amersham) according to the manufacturer’s protocol.

Kinase assays. Cells were lysed in lysis buffer. Cell lysates were clarifiedby centrifugation, and equal amounts of the lysate proteins (500 Ag) wereimmunoprecipitated with an antibody against the p85 subunit of PI3K or

p38 and conjugated to protein A/G plus-agarose (Santa Cruz Biotechnol-

ogy). In PI3K assay, the immune complexes were washed twice with PBS(pH 7.4) containing 1% NP40 and 1 mmol/L Na3VO4, twice with 100

mmol/L Tris-HCl (pH 7.5) containing 500 mmol/L LiCl and 1 mmol/L

Na3VO4, and twice with 50 mmol/L Tris-HCl (pH 7.2) containing

150 mmol/L NaCl. The kinase reactions were started by adding 5 mg/mLL-a-phosphatidylinositol (Sigma, St. Louis, MO) in 20 mmol/L HEPES

(pH 7.4), 5 mmol/L MnCl2, 10 Amol/L ATP, 5 ACi g-[32P]ATP, and2.5 mmol/L EGTA. After 20 minutes of incubation at room temperature,

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the reactions were quenched by adding 1 mol/L HCl. The phospholipidswere extracted using a 1:1 mixture of chloroform and methanol and

separated by TLC. Spots were visualized by autoradiography. In p38 kinase

assay, the beads were washed thrice with a solution [20 mmol/L Tris-HCl

(pH 7.5), 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L EDTA, and0.5% NP40] and once with a kinase assay buffer [50 mmol/L Tris-HCl

(pH 7.5), 137 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L Na3VO4,

2.5 mmol/L h-glycerophosphate, 2 mmol/L EDTA, and 5 Amol/L ATP] andthen subjected to kinase assay. p38 activities were measured in a reactionmixture consisting of a kinase assay buffer, 1 Ag activating transcriptionfactor-2 (ATF-2), and 5 ACi g-[32P]ATP for 20 minutes at 30jC. Thereaction was terminated by addition of SDS sample buffer, and the

samples were subjected to 10% SDS-PAGE. Phosphorylated ATF-2 wasvisualized by autoradiography.

In vitro invasion assays. Invasion assays were done using modified

Boyden chambers with polycarbonate Nucleopore membrane (Corning,Corning, NY) as described previously (25). Invasiveness was determined by

counting cells in five microscopic fields per well, and the extent of invasion

was expressed as an average number of cells per microscopic field.

Results

IR up-regulates MMP-2 secretion and invasion in functionalPTEN-deficient glioma cells. To investigate the role of IR in the

invasiveness of glioma cells, we first studied the effect of IR on thesecretion of MMPs and TIMPs in various glioma cell lines. BecauseIR did not trigger any cytotoxic effect on glioma cells after 24 hoursof irradiation with up to 10 Gy in serum-free condition, we used<10 Gy and 24 hours of incubation time throughout this study.Following IR (5 Gy) of different human and rat glioma cell lines,gelatin zymography of conditioned medium and Western blotanalysis of cell lysates showed significantly induced secretion andexpression of MMP-2, but not MMP-9, in some of the cell lines(Fig. 1A). Interestingly, IR-induced secretion of MMP-2 wasdetected in functional PTEN-deficient glioma cell lines (U87,U251, U373, and C6), whereas it was not detectable in glioma celllines harboring WT-PTEN (LN18 and LN428; Fig. 1A, top).Expression of TIMP-2, but not TIMP-1, was detected in all celllines but dominantly expressed in LN18 and LN428 cells and IR didnot affect the expression of TIMPs in these cells (Fig. 1A, bottom).We then did Matrigel invasion assays to test the effect of IR onglioma cells invasiveness. As shown in Fig. 1B , IR increased theinvasiveness of glioma cells lacking functional PTEN (U251 andU373) but not that of cells harboring WT-PTEN (LN18 and LN428).The IR-induced MMP-2 secretion, expression (Fig. 1C, top), andin vitro invasion (Fig. 1C, bottom) of glioma cells (U251 and U373)

Figure 1. Effect of IR on the secretion of MMP-2 and MMP-9 and invasion in various glioma cell lines. A, gelatin zymography analysis of serum-free conditionedmedium from various glioma cell lines (top ) and Western blot analysis of MMP-2, MMP-9, TIMP-1, and TIMP-2 in cell lysates from glioma cells (bottom ). Cells wereirradiated (5 Gy) and incubated in serum-free medium for 18 hours. Conditioned medium was collected and used for gelatin zymography. Cells were lysed in lysis bufferand subjected to Western blot for the detection of MMPs and TIMPs expression as described in Materials and Methods. B, Matrigel invasion assay of glioma cellsafter exposure to IR (5 Gy). Invasion through a layer of Matrigel was determined by a modified Boyden chamber method as described in Materials and Methods. Thecells invading through Matrigel were counted under microscope in five random fields at �200. C, gelatin zymography of conditioned medium and Western blot analysisof cell lysates (top ) and Matrigel invasion assay of U251 and U373 glioma cells (bottom ) after irradiation with various doses. D, Matrigel invasion assay of U251and U373 glioma cells after exposure to IR (5 Gy) in the presence or absence of synthetic MMP-2 inhibitor, OA-Hy. Each invasion data were obtained from threeseparate experiments. Columns, mean; bars, SD. *, 0.01 < P < 0.05; **, 0.005 < P < 0.01, versus control; @, 0.01 < P < 0.05; @@, 0.005 < P < 0.01, versus IR alone.Other experiments were done at least three independent conditions. Representative pictures.

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were dose dependent. In addition, IR-induced invasion wassuppressed by the addition of synthetic MMP-2 inhibitor, OA-Hy(Fig. 1D). Collectively, these results show that IR might induceinvasion in glioma cells harboring mutant PTEN through up-regulation of MMP-2 secretion, and we used U251 and U373 gliomacells for further experiments.IR-induced MMP-2 secretion in glioma cells is mediated at

the transcriptional level. Next, we did RT-PCR analysis todetermine whether the IR-induced increase of MMP-2 secretionwas controlled at the level of gene expressions. As shown in Fig. 2A ,IR (5 Gy) up-regulated transcription of MMP-2 mRNA in U251 andU373 glioma cells, and pretreatment with actinomycin D, but notcyclohexamide, blocked both the basal MMP-2 mRNA level andIR-induced MMP-2 up-regulation. Consistent with the results of our

RT-PCR analysis, a luciferase assay revealed that IR significantlyinduced MMP-2 promoter activity in U251 cells (Fig. 2B), indicatingthat IR up-regulates expression of the ECM-degrading protease,MMP-2, at the transcriptional level.PTEN suppresses IR-induced MMP-2 secretion in U251 and

U373 glioma cells. Because IR stimulated MMP-2 expression andsecretion in glioma cells lacking functional PTEN, we nextexamined the role of PTEN in this response. Gelatin zymographyshowed that transfection of WT-PTEN significantly reduced basalsecretion of MMP-2 as well as IR-induced MMP-2 up-regulation inU251 and U373 cell lines, whereas transfection of the PTEN mutantplasmid did not affect MMP-2 secretion (Fig. 3A). In addition,Western blot analysis showed that IR stimulated the phosphory-lation of Akt in both cells, whereas both basal and IR-stimulatedlevels of Akt phosphorylation were decreased in cells expressingWT-PTEN but not in cells expressing mutant PTEN (Fig. 3A).Because these data strongly suggested that the PI3K/Akt pathwayis critical for IR-induced MMP-2 secretion in these cells, we testedthe effect of specific inhibitors of these kinases on the IR-inducedsecretion of MMP-2. As shown in Fig. 3B , treatment of U251 andU373 cells with a specific inhibitor of PI3K (LY294002) or Akt(Akt inhibitor IV) significantly and dose-dependently blockedIR-stimulated secretion of MMP-2 and phosphorylation of Akt.To further confirm the involvement of the PI3K/Akt pathway, wetransfected plasmids encoding HA-tagged DN forms of p85 (DN-p85), the regulatory domain of PI3K, and Akt (DN-Akt) into U251and U373 cells, respectively. As shown in Fig. 3C , transfection ofDN-p85 and DN-Akt suppressed IR-induced MMP-2 secretion andAkt phosphorylation in these cells. We also investigated theinvolvement of mTOR, a downstream effector molecule of Akt, inIR-induced MMP-2 secretion using rapamycin, a specific inhibitorof mTOR. Figure 3D shows that IR significantly induced mTORphosphorylation, and pretreatment of U251 and U373 glioma cellswith rapamycin suppressed these events in a dose-dependentmanner. Taken together, these data strongly suggest that the PI3K/Akt/mTOR signaling pathway is required for IR-induced MMP-2secretion and that PTEN modulates these events in U251 and U373human glioma cells.Src-dependent EGFR phosphorylation is involved in IR-

induced MMP-2 secretion and Akt phosphorylation in U251and U373 glioma cells. Receptor tyrosine kinases (RTK), such asEGFR, are often activated in response to irradiation in variouscarcinoma cells and are typically overexpressed in glioma cells(34, 35). IR also can activate non-RTKs, such as Src, which cansignal downstream MAPK pathways in EGFR activation-dependentor activation-independent fashions (33, 35, 36). To investigate theinvolvement of RTKs in the IR-induced secretion of MMP-2, wepretreated U251 and U373 cells with various RTK inhibitors beforeIR and found that pretreatment of the glioma cells with specificinhibitors of EGFR (AG490 and AG1478) and a broad-spectrumRTK inhibitor (genistein), but not with specific inhibitor of ErbB2(AG825) or PDGFR (SU5402), significantly inhibited IR-inducedMMP-2 secretion and EGFR phosphorylation (Fig. 4A). To confirmthe involvement of EGFR in this event, we transduced adenovirusharboring WT-EGFR and found that, regardless of IR, over-expression of WT-EGFR significantly enhanced the secretion ofMMP-2 and phosphorylation of EGFR and Akt in both cell lines(Fig. 4B). On the contrary, however, pretreatment of these cellswith a neutralizing antibody against EGFR suppressed IR-inducedMMP-2 secretion and phosphorylation of EGFR and Akt (Fig. 4B),indicating that IR-induced EGFR activation is required for the

Figure 2. Effect of IR on the MMP-2 mRNA expression and MMP-2 promoteractivity in human glioma cells. A, RT-PCR analysis of MMP-2 mRNA expression.Total RNA was isolated after 12 hours of irradiation (5 Gy) from U251 andU373 cells pretreated with or without actinomycin D (ActD ; 10 Ag/mL) andcyclohexamide (CHX ; 10 Ag/mL) and subjected to RT-PCR. RT-PCR analysiswas done thrice independently. B, luciferase assay of MMP-2 promoter. U251cells were cotransfected with control reporter vector (pRL-CMV) or vectorcontaining MMP-2 promoter regions (pGL3-MMP-2) for 24 hours and irradiatedwith IR (5 Gy). After 6, 12, and 24 hours, respectively, the cells were lysed andthe extract was analyzed for luciferase activity. Each promoter activity datawere obtained from three separate experiments. Columns, mean; bars, SD.*, 0.01 < P < 0.05; **, 0.005 < P < 0.01.

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up-regulation of MMP-2 through the Akt signaling pathway. Wenext examined the possible involvement of Src kinase in theseevents. Figure 4C shows that IR induced phosphorylation of Srckinase, whereas transfection of DN-Src or pretreatment with aspecific inhibitor of Src (PP2) reduced IR-induced MMP-2 secretionand Src and Akt phosphorylation. In addition, PP2 completelyblocked EGFR phosphorylation induced by IR, whereas AG1478 didnot affect IR-induced phosphorylation of Src (Fig. 4D), indicatingthat Src might be an upstream kinase of the EGFR/Akt signalingpathway in irradiated U251 and U373 glioma cells. Taken together,these data strongly suggest that IR-induced MMP-2 secretion isaccomplished by Src/EGFR-mediated Akt signal transduction.p38 is also involved in IR-induced Akt phosphorylation and

MMP-2 secretion in U251 and U373 glioma cells. To furtherunderstand the mechanism of IR-mediated induction of MMP-2secretion, we next used specific MAPK inhibitors to examine theroles of three MAPKs. Gelatin zymography showed that SB203580, aspecific inhibitor of p38, but not PD98059 and SP600125, specificinhibitors of ERK1/2 and JNK, respectively, significantly abolishedboth basal and IR-mediated MMP-2 secretion in U251 and U373human glioma cells (Fig. 5A). To confirm the involvement of p38 inIR-inducedMMP-2 expression, we transiently infected both cell lineswith retrovirus-mediated WTand DN-p38 expression constructs. Asexpected, infection with DN-p38 markedly reduced the basal and

IR-induced secretion of MMP-2 in both cell lines (Fig. 5B). Incontrast, introduction of WT-p38 significantly enhanced the basalsecretion of MMP-2, and irradiation further increased this MMP-2secretion (Fig. 5B). Interestingly, introduction of DN-p38 signifi-cantly suppressed both basal and IR-induced Akt phosphorylationlevels, whereas WT-p38 up-regulated Akt phosphorylation (Fig. 5B).To further confirm this effect, we transiently transfected the cellswith a plasmid encoding WT-MKK6, an upstream kinase of p38, andfound that expression of WT-MKK6 enhanced MMP-2 secretion,increased the activation of ATF-2 (a downstream target molecule ofp38), and increased the basal and IR-induced phosphorylation levelsof Akt (Fig. 5C). Furthermore, pretreatment of cells with AG1478 andPP2, but not with LY294002, significantly suppressed IR-inducedp38 kinase activity (Fig. 5D, top). Finally, a PI3K assay revealedthat treatment of cells with AG1478 and PP2, but not SB203580,significantly suppressed basal and IR-induced PI3K activity(Fig. 5D, bottom). Collectively, these findings indicate that Src/EGFR-mediatedMKK6/p38 activation is required for IR-induced Aktactivation and subsequent MMP-2 secretion in U251 and U373glioma cells independent of PI3K-mediated activation of Akt.Specific inhibitors of Src, EGFR, p38, PI3K, and Akt suppress

IR-induced invasion of U251 and U373 glioma cells. Becauseour results revealed that the Src, EGFR, p38, PI3K, and Akt kinasesare critical for IR-induced MMP-2 up-regulation in U251 and U373

Figure 3. Involvement of PI3K/Akt/mTOR signaling pathway in IR-induced MMP-2 expression of glioma cells. Gelatin zymography of conditioned medium and Westernblot of cell lysates from U251 and U373 cells transfected with WT-PTEN (PTEN) or mutant PTEN (C124S; A), DN-p85 or DN-Akt (C ), and control vector (pCDNA3)and treated with specific inhibitor of PI3K (LY294002), Akt (Akt inhibitor IV; B), or mTOR (rapamycin; D ). Cells were irradiated (5 Gy) and incubated in serum-freemedium for 18 hours. Conditioned medium was collected and subjected to gelatin zymography for the detection of secreted MMP-2, and cell extracts (50 Ag each)were resolved by SDS-PAGE and probed with antibodies against phosphorylated Akt (A-C ) or phosphorylated mTOR (D ). To verify equal loading or expression ofDN constructs, the blots were probed with antibodies against Akt (A-C ), HA (C ), or mTOR (D ). All experiments were done at least thrice independently. Detailedexperimental procedures are described in Materials and Methods.

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cells, we next examined whether the inhibition of these path-ways could suppress the IR-induced invasion by these cells. Asexpected, treatment of U251 and U373 cells with PP2, AG1478,SB203580, LY294002, and Akt inhibitor IV, but not PD98059 orSP600125, ameliorated the IR-induced invasiveness of U251 andU373 glioma cells (Fig. 6A). These data strongly indicate that thesesignaling pathways are important for IR-induced MMP-2 up-regulation and increased glioma cell invasiveness. Therefore, it ishighly possible that Src/EGFR-mediated p38/Akt and PI3K/Aktsignaling pathways play a key role in IR-induced MMP-2 expressionand subsequent invasiveness in U251 and U373 glioma cells (Fig. 6B).

Discussion

Despite aggressive treatment strategies, the prognosis ofglioblastoma remains poor partly due to its high invasiveness intoadjacent brain tissues. Here, we examined the regulatorymechanism of IR in MMP-2 secretion and invasion of glioma cells.We observed that IR enhanced glioma cell invasiveness and thatthis effect was at least partly mediated by increased MMP-2

expression in PTEN mutated glioma cell lines. Furthermore, IR wasfound to activate the PI3K/Akt and p38/Akt signaling pathways inU251 and U373 cells, with Src-mediated EGFR phosphorylationacting upstream of these pathways. Collectively, these findingsseem to identify candidate signaling pathways that may be used aspotential targets for controlling IR-induced glioma invasiveness.The IR-induced invasiveness of PTEN mutant glioma cells

required up-regulation of MMP-2. Elevated levels of MMP-2 havebeen reported in malignant gliomas (6–12); most glioma cellsconstitutively express MMP-2, and its expression is correlated withthe invasive potential of these tumor cells (11–14, 24, 37, 38). Similarto other members of the metalloproteinase family, the synthesisand function of MMP-2 can be regulated by various factors, not allof which have been fully elucidated. Transforming growth factor-h(39), laminin, an ECM component (40), interleukin-8 (41), andinsulin like growth factor-1 (42) have all been shown to alter theexpression or function of MMP-2. In addition, IR potently inducesMMP-2 in various normal cells (43–45) and was also found toinduce invasion and metastasis in several types of cancer cellsin vitro and in vivo , including gliomas (28–31).

Figure 4. Involvement of Src-dependent EGFR phosphorylation in the MMP-2 expression and Akt phosphorylation of glioma cells. Gelatin zymography of conditionedmedium and/or Western blot of cell lysates from U251 and U373 cells treated with specific inhibitors of EGFR (AG490 and AG1478), ErbB2 (AG825), PDGFR (SU5402),and broad-spectrum inhibitor (genistein; A), transduced with control adenovirus (Ad-LacZ ) or adenoviral WT-EGFR (Ad-wt-EGFR ) and treated with controlimmunoglobulin G (IgG ) or neutralizing antibody against EGFR (B), transfected with control vector (pcDNA) or DN-Src and treated with specific inhibitor of Src(PP2; 10 Amol/L; C ), and treated with AG1478 (5 Amol/L) and PP2 (D ). Cells were irradiated (5 Gy) and incubated in serum-free medium for 18 hours. Conditionedmedium was collected and subjected to gelatin zymography for the detection of secreted MMP-2, and cell extracts (50 Ag each) were resolved by SDS-PAGEand probed with antibodies against phosphorylated EGFR (A, B , and D ), phosphorylated Akt (B and C ), or phosphorylated Src (C and D ). To verify equal loading,the blots were probed with antibodies against EGFR (A, B , and D ), Akt (A-C ), Src (C and D), or h-actin (C ). All experiments were done at least thrice independently.Detailed experimental procedures are described in Materials and Methods.

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Here, we found that IR potently induced MMP-2 expression andinvasion in various glioma cells. Interestingly, IR-induced MMP-2expression and invasion were restricted to cells lacking functionalPTEN, and transfection of WT-PTEN, but not mutant PTEN, inthese cells resulted in the suppression of these events. Thesefindings strongly indicate the involvement of PI3K/Akt signalingpathway in IR-induced MMP-2 expression and invasion of gliomacells. Indeed, IR-activated PI3K/Akt pathway, treatment of specificinhibitors, or transfection of DN mutant forms of these kinasessignificantly reduced these events. In some ways, these findings arecontradictory to the report of Wild-Bode et al., who reported thatsublethal doses of irradiation up-regulated expression of MMP-2and MT1-MMP, down-regulated expression of TIMP-2, andincreased invasiveness in glioma cells (U87, LN229, and LN18)regardless of PTEN and p53 status (28). However, our finding isconsistent with that of Cordes et al., who reported that IR did not

affect invasion of LN-229 and LN-18 cells harboring WT-PTEN (32).These authors also found that MMP-2 and MT1-MMP were up-regulated and TIMP-2 was down-regulated in irradiated A172 andU138 cells having no PTEN protein; however, IR suppressed or didnot affect invasion of these cells (32). In the present study, wefound that a large amount of TIMPs expression was detected inglioma cell lines harboring WT-PTEN but not in cells with mutantPTEN (Fig. 1B), and IR did not affect the secretion of TIMPs.Despite the large amount of MMP-2 expression, the basal invasiveactivity of WT-PTEN harboring cells was comparatively weakerthan the cells with mutant PTEN, which might possibly be due tothe concomitant expression of endogenous MMP inhibitors, TIMP-1 and TIMP-2. On the contrary, gelatin zymography showed highMMP-2 activity in LN18 and LN428 cells, which might be due to thedissociation of TIMP-2 by SDS on the gel during the process of thezymography as described in Materials and Methods. In addition,

Figure 5. Involvement of MKK6/p38 pathway in the MMP-2 secretion and Akt phosphorylation of glioma cells. A, gelatin zymography of conditioned medium fromU251 and U373 glioma cells treated with specific inhibitors of three MAPKs: p38 (SB203580), ERK1/2 (PD98059), and JNK (SP600125). B, gelatin zymographyof conditioned medium and Western blot of cell lysates from U251 and U373 glioma cells transduced with control retrovirus (MFG ), retroviral DN-p38 (Rt-DN-p38 ),or retroviral WT-p38 (Rt-WT-p38 ). C, gelatin zymography of conditioned medium and Western blot analysis and immunocomplex kinase assay of cell lysates fromU251 and U373 glioma cells transfected with control vector (pcDNA) or WT-MKK6 plasmid. D, immunocomplex kinase assay of p38 (top ) and PI3K (bottom ) in U251and U373 glioma cells treated with AG1478 (5 Amol/L), PP2 (10 Amol/L), LY294002 (10 Amol/L), or SB203580 (5 Amol/L). Cells were irradiated (5 Gy) and incubatedin serum-free medium for 18 hours. Conditioned medium was collected and subjected to gelatin zymography for the detection of secreted MMP-2 (A) and cell extracts(50 Ag each) were resolved by SDS-PAGE and probed with antibodies against phosphorylated p38 and p38 (B), phosphorylated Akt, and Akt (B and C). ATF-2and L-a-phosphatidylinositol reacted with immunoprecipitates of p38 and PI3K, respectively, were resolved by SDS-PAGE (for p38; C and D ) or TLC (for PI3K; D )and subsequently autoradiographed. PIP is the phosphorylated form of L-a-phosphatidylinositol. To verify equal loading, the blots were probed with antibodies againsth-actin (B ) and/or Akt (B and C). All experiments were done at least thrice independently. Detailed experimental procedures are described in Materials and Methods.

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our previous study showed that introduction of WT-PTENenhances TIMP-1 and TIMP-2 expressions in glioma cells lackingfunctional PTEN (25). Therefore, the present and earlier studiesshow some differences. These are most likely due to differentexperimental conditions and/or different cellular states; neverthe-less, it is clear that IR enhances the invasion of glioma cells andMMP-2 might be a critical factor in this event and that PI3K/Aktsignaling pathway is involved in this event, partly at least.Another critical factor in glioma invasion seems to be EGFR,

which is overexpressed in up to 50% of malignant glioma cells (35).When bound by its ligands (e.g., epidermal growth factor andtransforming growth factor-a), EGFR is activated and triggersdownstream signaling cascades. In addition, EGFR may beactivated by irradiation in various cancer cells, including glioma(33–35), and this effect is believed to be a major cause ofradioresistance in glioma cells. Our data showed that IR potentlyinduced EGFR phosphorylation and that blockade of EGFRactivation by specific inhibitors and neutralizing antibodieseffectively suppressed IR-induced MMP-2 expression and Aktphosphorylation. UV irradiation-mediated EGFR phosphorylationhas recently been reported to depend on activation of Src, a non-RTK (36), suggesting that activation of Src may also mediate IR-induced EGFR activation. Consistent with this hypothesis, our datashowed clearly that IR induced Src phosphorylation in glioma cellsand that inhibition of Src phosphorylation by a specific inhibitor(PP2) or DN-Src suppressed IR-induced EGFR phosphorylation,PI3K activation, Akt phosphorylation, and MMP-2 expression.These results indicate that IR-induced MMP-2 expression is most

likely accomplished at least partially through Src/EGFR-mediatedPI3K/Akt signaling.Because IR seems to preferentially transmit signals via EGFR to

MAPK family members (i.e., ERK1/2, p38, and JNK; ref. 33), weexamined the involvement of MAPKs in IR-induced MMP-2expression and invasion. Of the specific MAPK inhibitors tested,only the p38 inhibitor inhibited IR-induced MMP-2 expression inU373 and U251 glioma cells. Furthermore, we found that IRactivated p38 and introduction of DN-p38 blocked IR-inducedMMP-2 expression, whereas introduction of WT-MKK6 (anupstream kinase of p38) or WT-p38 enhanced both basal andIR-induced MMP-2 expression. IR-induced activation of p38 wassignificantly blocked by Src and EGFR inhibitors and DN-Src,indicating that p38 acts as an another downstream targetmolecule of IR-induced Src/EGFR activation. Interestingly, block-ade of p38 effectively suppressed IR-induced Akt phosphorylationbut did not affect PI3K activity. In addition, PI3K activity did notaffect IR-induced p38 kinase activity. Several studies have shownthat p38 regulates Akt phosphorylation in a PI3K-dependent orPI3K-independent fashion under various experimental situations(46, 47). Therefore, p38 and PI3K seem to be another importantkinases that signal to Akt pathway in glioma cells invasion, partlyat least.Although IR-induced MMP-2 expression and invasion were

effectively controlled by the above-mentioned kinases, thesesignaling molecules were also found to be involved in basalMMP-2 expression and invasion of glioma cells. Furthermore,inhibition of these kinases did not completely reduce the

Figure 6. Matrigel invasion assay of U251 andU373 cells after exposure to irradiation (5Gy).A, cells were pretreated with various kinase inhibitors and loaded onto upperchamber coated with Matrigel. Detailed experimental procedures are described in Materials and Methods. The cells invading through Matrigel were counted undermicroscope in five random fields at�200. Representative of three separate experiments.Columns, mean; bars, SD. *, 0.01 <P < 0.05; **, 0.005 <P < 0.01, versus control;@, 0.01 < P < 0.05; @@, 0.005 < P < 0.01, versus IR alone. B, proposed model for signaling pathways involved in the IR-induced invasion of glioma cells.

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IR-induced MMP-2 expression and invasion of glioma cells.Therefore, it is highly likely that these signaling molecules arenot the only ones responsible for IR-induced MMP-2 expressionand invasion of glioma cells. Further studies are needed to uncoverother critical signaling molecules involved in these events.In summary, our results indicate that IR induces Src-dependent

EGFR activation, which triggers the p38/Akt and PI3K/Aktsignaling pathways, leading to increased MMP-2 expression andheightened invasiveness of mutant PTEN glioma cells. Althoughfuture work will be required to elucidate whether this signalingoccurs in vivo , these findings provide possible new targets for

controlling the invasiveness of IR-treated glioma cells, perhapsfacilitating a strategy for preventing radioresistance in this deadlycancer.

Acknowledgments

Received 12/5/2005; revised 6/19/2006; accepted 6/27/2006.Grant support: National Nuclear R&D Program of Ministry of Science and

Technology (Seoul, Korea).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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

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2006;66:8511-8519. Cancer Res   Chang-Min Park, Myung-Jin Park, Hee-Jin Kwak, et al.   and Phosphatidylinositol 3-Kinase/Akt Signaling Pathways

Mediated p38/Akt−Src/Epidermal Growth Factor Receptor Secretion and Invasion of Glioma Cells through

Ionizing Radiation Enhances Matrix Metalloproteinase-2

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