[CANCER RESEARCH 62, 6318 – 6322, November 1, 2002] PTEN Suppresses Hyaluronic Acid-induced Matrix Metalloproteinase-9 Expression in U87MG Glioblastoma Cells through Focal Adhesion Kinase Dephosphorylation 1 Myung-Jin Park, Mi-Suk Kim, In-Chul Park, Hee-Seok Kang, Heon Yoo, Seok Hee Park, Chang Hun Rhee, Seok-Il Hong, and Seung-Hoon Lee 2 Laboratory of Cell Biology [M-J. P., I-C. P., C. H. R., H-S. K., S-I. H., S-H. L], Korea Cancer Center Hospital, Seoul 139-706, Korea, and Neurooncology Clinic [M-S. K., H. Y., S-H. L.], Division of Basic Science [S. H. P.], National Cancer Center, Goyang, Gyeonggi, 411-351, Korea ABSTRACT Glioblastoma is a severe type of primary brain tumor and its invasion is strongly correlated with the secretion of matrix metalloproteinases (MMPs). To investigate a role of PTEN, a tumor suppressor gene, in the regulation of hyaluronic acid (HA)-induced invasion of glioma cells, we examined the secretion of MMP-9 in various glioma cells with or without a functional PTEN gene. The secretion of MMP-9 in glioma cells lacking functional PTEN (U87MG, U251MG, and U373MG) was induced by HA, although not in wildtype (wt)-PTEN-harboring cells (LN229, LN18, and LN428). In addition, stable expression of wt-PTEN into U87MG cells significantly decreased the secretion of HA-induced MMP-9 and basal levels of MMP-2, inhibiting the activation of focal adhesion kinase and extracellular signal-regulated kinase 1/2, whereas the secretion levels of the tissue inhibitor of metalloproteinase-1 and -2 were increased, finally resulting in the inhibition of invasion by HA in vitro. Ectopic expressions of adenoviral (Ad)-wt-PTEN and -lipid phosphatase-deficient (G129E)- PTEN, but not both protein and -lipid phosphatase-deficient (C124S)- PTEN, reduced MMP-9 secretion and invasion by HA. These results were also confirmed by expressions of Ad-wt-PTEN and Ad-G129E-PTEN in other glioblastoma cells lacking functional PTEN, U251MG, and U373MG. These findings strongly suggest the possibility that PTEN may block HA-induced MMP-9 secretion and invasion through its protein phosphatase activity. INTRODUCTION Glioblastoma, a severe type of primary brain tumor, is lethal because of local invasion into brain parenchyma. Glioma invasion is strongly correlated with the secretion of MMPs 3 (1, 2). MMPs are a large family of zinc-dependent neutral endopeptidases, and are in- volved in the degradation of many different components of the extra- cellular matrix. Among the MMPs, MMP-9 specifically targets type IV collagen, a major component of the basement membrane, and appears to play a crucial role in glioma invasion across this barrier (3). HA is the principal glycosaminoglycan found in extracellular ma- trix of brain. It binds to cell-surface receptors such as CD44 and appears to be involved in cell adhesion, migration, proliferation, and tumor progression (4, 5). In human brain, HA is distributed in white matter fiber tracts, which form the most frequent route of glioma dissemination (6). There are several studies that suggest an important role of HA in glioma cell invasion in vitro (7, 8). However, there have been only few studies on the molecular mechanism of HA-mediated invasion of glioma cells (9). Furthermore, the specific role of HA on the secretion of MMPs and the invasion by glioma cells is not well understood. PTEN (also called MMAC1) is a tumor-suppressor gene located on human chromosome 10q23.3 (10, 11), and it regulates cell growth, apoptosis (12), and interaction with the extracellular matrix, and inhibits cell migration, spreading, and focal adhesion (13). PTEN protein exhibits dual specificity protein phosphatase activity in vitro (14) and its potential cellular target of FAK (15). However, it can also dephosphorylate the lipid signal transduction molecules phosphatidy- linositol 3,4,5-triphosphate and phosphatidylinositol 3,4-bisphos- phate, which are both involved in the PI3K pathway (14). It has been suggested that dephosphorylation of FAK by the PTEN protein is correlated with cell spreading, migration, and invasion (13, 15). In addition, Koul et al. (16) recently reported that PTEN suppresses MMP-2 gene expression, and invasion of glioma cells and phospha- tase activity is essential in these events. On the other hand, it has also been reported that the lipid phosphatase activity of PTEN is not required in the invasive potential of glioma cells (17). In this report, we describe the effect of HA on the secretion of MMP-9 and invasion of human glioma cell lines, and the role of PTEN in these events. Our findings suggest that PTEN suppresses HA-induced secretion of MMP-9, possibly through FAK dephospho- rylation; thus, PTEN inhibits HA-induced invasion via control of MMP-9 secretion. MATERIALS AND METHODS Reagents and Cell Culture. HA was obtained from Sigma Chemical Co. (St. Louis, MO) and reconstituted in DMEM (Life Technologies, Inc., Grand Island, NY). The following inhibitors purchased from Calbiochem (La Jolla, CA) were used in this study: Ras-specific inhibitor (damnacanthal), PKC inhibitor (G06983 and GF109203X), MAPK inhibitor (PD98059) and ERK- 1/2 inhibitor (SB203580). Human glioma cell lines [U87MG, U251MG, and U373MG (obtained from American Type Culture Collection) and LN18, LN428, and LN229 (a generous gift from Dr. Frank Funari, Ludwig Institute for Cancer Research, La Jolla, CA)] were maintained in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 units/ml) and streptomycin (100 g/ml) at 37°C in a humidified atmosphere containing 5% CO 2 . Plasmid Construction and Cell Transfection. wt-PTEN cDNA was ob- tained from Dr. Hong Sun (Yale University, New Haven, CT) and cloned into pcDNA3 vector to generate pcDNA3-PTEN. The entire open reading frame was sequenced to confirm the correct sequence. U87MG cells were transfected with pcDNA3-PTEN or pcDNA3 (no insert) in triplicate dishes and in three independent experiments by using the Effectene reagent (Qiagen, Valencia, CA) according to the manufacturer’s protocol. After 24 h, cells were split at a 1:5 dilution and exposed for 2–3 weeks in G418 (Boehringer Mannheim, Indianapolis, IN)-containing medium (800 g/ml) and colonies were picked for their resistance to G418. Expression of PTEN was confirmed by Western blot analysis using a monoclonal antibody against PTEN. Construction and Infection of Recombinant Adenovirus. To construct Ad-PTEN vectors, 1.2-kb fragments of PTEN cDNAs [wt- and mutant type- PTEN (C124S and G129E, a generous gift from Dr. Young E. Whang, University of North Carolina School of Medicine, Chapel Hill, NC)] were cloned into the KpnI and XhoI sites of pShuttle-CMV vector (AdEasy Adeno- viral Vector System; Stratagene, La Jolla, CA) and cotransformed into Esch- erichia coli BJ5183 cells with the pAdEasy-1 vector, using the electroporation Received 3/5/02; accepted 8/26/02. 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. 1 Supported by the National Cancer Control Program of the Ministry of Health and Welfare and the National Nuclear R & D program of the Ministry of Science and Technology, Seoul, Korea. 2 To whom requests for reprints should be addressed, at Neurooncology Clinic, National Cancer Center, 809 Madu-dong, Ilsan-gu, Goyang, Gyeonggi, 411-351, Korea. Phone: 82-31-920-1660; Fax: 82-31-920-1520; E-mail: nslsh@ncc.re.kr. 3 The abbreviations used are: MMP, matrix metalloproteinase; HA, hyaluronic acid; FAK, focal adhesion kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; ERK 1/2, extracellular signal-regulated kinase 1/2; TIMP, tissue inhibitor of metalloproteinase; wt, wild type; Ad, adenoviral; HEK, human embryonic kidney. 6318 Research. on November 9, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from