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Originally published In Press as doi:10.1074/jbc.M601803200 on April 19, 2006

J. Biol. Chem., Vol. 281, Issue 25, 17492-17500, June 23, 2006
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Autotaxin Is Overexpressed in Glioblastoma Multiforme and Contributes to Cell Motility of Glioblastoma by Converting Lysophosphatidylcholine TO Lysophosphatidic Acid*

Yasuhiro Kishi{ddagger}, Shinichi Okudaira{ddagger}, Masayuki Tanaka{ddagger}, Kotaro Hama{ddagger}, Dai Shida§, Joji Kitayama§, Takao Yamori, Junken Aoki{ddagger}1, Takamitsu Fujimaki||, and Hiroyuki Arai{ddagger}

From the {ddagger}Graduate School of Pharmaceutical Sciences, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the §Graduate School of Medicine, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo 170-8455, and the ||Department of Neurosurgery, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo 173-8605, Japan

Received for publication, February 24, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autotaxin (ATX) is a multifunctional phosphodiesterase originally isolated from melanoma cells as a potent cell motility-stimulating factor. ATX is identical to lysophospholipase D, which produces a bioactive phospholipid, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC). Although enhanced expression of ATX in various tumor tissues has been repeatedly demonstrated, and thus, ATX is implicated in progression of tumor, the precise role of ATX expressed by tumor cells was unclear. In this study, we found that ATX is highly expressed in glioblastoma multiforme (GBM), the most malignant glioma due to its high infiltration into the normal brain parenchyma, but not in tissues from other brain tumors. In addition, LPA1, an LPA receptor responsible for LPA-driven cell motility, is predominantly expressed in GBM. One of the glioblastomas that showed the highest ATX expression (SNB-78), as well as ATX-stable transfectants, showed LPA1-dependent cell migration in response to LPA in both Boyden chamber and wound healing assays. Interestingly these ATX-expressing cells also showed chemotactic response to LPC. In addition, knockdown of the ATX level using small interfering RNA technique in SNB-78 cells suppressed their migratory response to LPC. These results suggest that the autocrine production of LPA by cancer cell-derived ATX and exogenously supplied LPC contribute to the invasiveness of cancer cells and that LPA1, ATX, and LPC-producing enzymes are potential targets for cancer therapy, including GBM.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autotaxin (ATX)2 is a 125-kDa glycoprotein and a potent tumor cell motogen that was originally isolated from the conditioned medium of A2058 human melanoma cells as a cell motility-stimulating factor for melanoma cells (1). ATX was subsequently identified as a member of a family of ecto/exoenzymes referred to as nucleotide pyrophosphatases/phosphodiesterases (NPPs) (2, 3). The three cloned members of this family (PC-1/NPP1, ATX/NPP2, and B-10/NPP3) share a 47-55% amino acid sequence identity. PC-1/NPP1 and B-10/NPP3 hydrolyze 5'-phosphodiester bonds in nucleotides in vitro, whereas ATX/NPP2 shows only weak activity at hydrolyzing such bonds. ATX is synthesized as a type II membrane protein and is released from cells in a soluble form by an unknown mechanism (3, 4). Enhanced expression of ATX in Ras-transformed NIH3T3 cells greatly enhances their invasive, tumorigenic, and metastatic potentials (5). In addition, enhanced expression of ATX has been repeatedly demonstrated in various malignant tumor tissues including non-small cell lung cancer (6), breast cancer (7, 8), renal cell cancer (9), hepatocellular carcinoma (10, 11), and thyroid cancer (12), suggesting that ATX confers the tumorigenic and metastatic potentials of cancer cells. However, there is no direct evidence to show such a hypothesis so far.

The mechanism by which ATX exhibits its biological activity toward various cancer cells was unknown. An ATX point mutant that is deficient in 5'-nucleotide phosphodiesterase activity was found to abolish the cell motility-stimulating activity of ATX (13), indicating that the migratory response to ATX requires an intact catalytic site. Recently, we and others showed that ATX has lysophospholipase D (lysoPLD) activity, which catalyzes a reaction to produce a bioactive lysophospholipid, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC) (14, 15). ATX has a significantly lower Km for LPC than the Km for the classical nucleotide substrate. Because LPA has long been defined as a cell motility-stimulating factor for various cell types including glioblastomas (16-18), ATX has been suggested to regulate motility by producing LPA through the G-protein-coupled receptor. Indeed, recent studies have shown that ATX stimulates the cell motility of various cancer cells in vitro through one of the LPA receptors, LPA1 (19-22). Taking account of the fact that elevated ATX expression has been detected in various tumors (6-12), it is possible that certain cancer cells utilize the ATX-LPC-LPA-LPA1 system for their motility. In these cells, a possible regulatory factor that remains to be characterized is LPC. LPC is always present in plasma. In human plasma, its concentration ranges from 100 to 300 µM. LPC is also detected in other biological fluids such as seminal fluids and cerebrospinal fluids and in tissues and various types of cells but at much lower concentrations than in plasma (23, 24).

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. Removal of the tumor mass transiently improves the condition of the patient, but the ability of GBM cells to infiltrate normal brain tissue invariably almost always leads to tumor recurrence. Thus, most patients experience recurrence within 1 year (25), and less than 20% of the patients survive more than 2 years (26). GBM cells (glioblastomas) are highly motile and invade the normal brain parenchyma diffusely (27). Several factors responsible for their invasive phenotype have been reported, such as certain extracellular matrix proteins including laminin, fibronectin, and/or collagen can promote glioma cell migration (28, 29). Secreted matrix metalloproteinases remodel the extracellular matrix, creating pathways more conducive to migration through normal brain tissue (30). Investigations of the factors that affect the motility of glioblastomas are of particular interest because an understanding of these factors is needed for valid GBM therapy. Because the blood-brain barrier (BBB) is disrupted in GBM tissue, some components in plasma might affect the cell motility of glioblastomas (31, 32). In this study, we examined the expression of ATX and LPA1 in brain tumor tissues and various tumor cell lines and found that both ATX and LPA1 are predominantly expressed in glioblastomas and GBM tissue. Using glioblastomas as a model system, we evaluated the effects of ATX, LPA1, LPA, and LPC on the motility of the cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—1-oleoyl-LPA (18:1) and 1-oleoyl-LPC (18:1) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Ki16425 was kindly provided by Dr. Hideo Ohata (Kirin Brewery Co., Takasaki, Japan).

Cell Lines—All human tumor cell lines were maintained in RPMI 1640 (Sigma) supplemented with 2 mM glutamine, 1x penicillin/streptomycin, and 5% (v/v) heat-inactivated fetal bovine serum as described previously (33). The cell lines used in this study were NCl-H23 (lung), NCl-H226 (lung), NCl-H522 (lung), NCl-H460 (lung), A549 (lung), DMS273 (lung), DMS114 (lung), HCC-2998 (colon), HT-29 (colon), WiDr (colon), HCT-15 (colon), DLD1 (colon), SW480 (colon), LOVO (colon), CaRI (rectum), WiDr (colon), CaCo2 (colon), Colo320 (colon), Colo201 (colon), HCT-116 (colon), KM12 (colon), HT1080 (colon), RXF-631L (renal), ACHN (renal), OVCAR3 (ovary), OVCAR4 (ovary), OVCAR5 (ovary), OVCAR8 (ovary), SKOV-3 (ovary), U251 (CNS), SF295 (CNS), SF539 (CNS), SF268 (CNS), SNB75 (CNS), SNB78 (CNS), MKN45 (stomach), MKN28 (stomach), St4 (stomach), MKN1 (stomach), MKN7 (Stomach), MKN74 (stomach), KatoIII (stomach), MKN28 (stomach), MKN45 (stomach), MKN74 (stomach), MDA-MB231 (breast), HBC4(breast), BSY1 (breast), MCF7 (breast), DU145 (prostate), PC3 (prostate), HPC5 (others), A2058 (melanoma), and HeLa (uterine cervix). Mouse 203G glioma cells were maintained in RPMI 1640 supplemented with 2 mM glutamine, 1x penicillin/streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum. This cell line was kindly provided by Dr. Koji Adachi (Nippon Medical School Tokyo, Japan).

Quantitative Real-time RT-PCR—From various cancer tissues and cancer cell lines, total RNA from cells was isolated using ISOGEN (Nippongene, Toyama, Japan) and reverse-transcribed using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen). Oligonucleotide primers for PCR were designed using Primer Express Software (Applied Biosystems, Foster City, CA). The sequences of the oligonucleotides used in PCR were as follows: ATX (human), forward, 5'-GGGTGAAAGCTGGAACATTCTT-3'; ATX (human), reverse, 5'-GCCACCGCAATATGGAATTATAAG-3'; LPA1 (human), forward, 5'-AATCGGGATACCATGATGAGTCTT-3'; LPA1 (human), reverse, 5'-CCAGGAGTCCAGCAGATGATAAA-3'; LPA2 (human), forward, 5'-CGCTCAGCCTGGTCAAGACT-3'; LPA2 (human), reverse, 5'-TTGCAGGACTCACAGCCTAAAC-3'; LPA3 (human), forward, 5'-AGGACACCCATGAAGCTAATGAA-3'; LPA3 (human), reverse, 5'-GCCGTCGAGGAGCAGAAC-3'; LPA4 (human), forward, 5'-CCTAGTCCTCAGTGGCGGTATT-3'; LPA4 (human), reverse, 5'-CCTTCAAAGCAGGTGGTGGTT-3'; ATX (mouse), forward, 5'-GGAGAATCACACTGGGTAGATGATG-3'; ATX (mouse), reverse, 5'-ACGGAGGGCGGACAAAC-3'; LPA1 (mouse), forward, 5'-GAGGAATCGGGACACCATGAT-3'; LPA1 (mouse), reverse, 5'-ACATCCAGCAATAACAAGACCAATC-3'; LPA2 (mouse), forward, 5'-GACCACACTCAGCCTAGTCAAGAC-3'; LPA2 (mouse), reverse, 5'-CTTACAGTCCAGGCCATCCA-3'; LPA3 (mouse), forward, 5'-GCTCCCATGAAGCTAATGAAGACA-3'; LPA3 (mouse), reverse, 5'-AGGCCGTCCAGCAGCAGA-3'; LPA4 (mouse), forward, 5'-CAGTGCCTCCCTGTTTGTCTTC-3'; LPA4 (mouse), reverse, 5'-GAGAGGGCCAGGTTGGTGAT-3'; GAPDH (human/mouse), forward, 5'-GCCAAGGTCATCCATGACAACT-3'; GAPDH (human/mouse), reverse, 5'-GAGGGGCCATCCACAGTCTT.

PCR reactions were performed using an ABI Prism 7000 sequence detection system (Applied Biosystems). The transcript number of human GAPDH was quantified, and each sample was normalized on the basis of GAPDH content.

LysoPLD Assay—Samples were incubated with 2 mM LPC (14:0) in the presence of 100 mM Tris-HCl (pH 9.0), 500 mM NaCl, 5 mM MgCl2, and 0.05% Triton X-100 during indicated hours at 37 °C. The liberated choline was detected by an enzymatic photometric method as described (14). Briefly, the liberated choline was oxidized by choline oxidase, and the hydrogen peroxide generated was quantified using horseradish peroxidase and TOOS reagent (N-ethyl-N-(2-hydroxy-3-sulfoproryl)-3-methylanikine, Dojin, Tokyo, Japan).

SDS-PAGE/Western Blotting—Both cells and cell media were used to test for ATX protein expression. Cells were homogenized in phosphate-buffered saline, and then the cell homogenates were separated into membrane and soluble fractions by centrifugation at 100,000 x g for 60 min. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes using the Bio-Rad protein transfer system. The membranes were blocked with 10 mM Tris-HCl (pH 7.5) containing 150 mM sodium chloride, 5% (w/v) skimmed milk, and 0.05% (v/v) Tween 20, incubated with anti-lysoPLD monoclonal antibody (3D1) (23), and then treated with anti-rat IgG-horseradish peroxidase. Proteins bound to the antibody were visualized with an enhanced chemiluminescence kit (ECL, Amersham Biosciences).

Isolation of Stable ATX Transfectant—The plasmid vector pCAGGS (kindly provided by Dr. Junichi Miyazaki (34)) was utilized to create rat ATX (ATX-t) tagged with the Myc epitope at the C terminus (pCAGGS-rATX-Myc). To establish stable transfectants, 203G glioma cells were transfected with pCAGGS-rATX-Myc using Lipofectamine Plus reagents (Invitrogen) as recommended by the manufacturer and were grown in RPMI 1640 containing 800 µg/ml G418 (Wako). Individual G418-resistant clones were isolated by limiting dilution and screened by immunocytochemistry using Myc antibody and by measuring the lysoPLD activities of the culture media.

Recombinant ATX Preparations—Rat ATX was expressed and partially purified using baculovirus system as described previously (14). The purified ATX was dialyzed in phosphate-buffered saline and used for cell motility assays.

Boyden Chamber Assay—Chemotaxis was assayed as described previously (21). In brief, polycarbonate filters with 8-µm pores (Neuro-Probe, Inc., Gaithersburg, MD) were coated with 0.001% fibronectin (Sigma). Cells (1 x 105 cells in 200 µl/well) were loaded into the upper chambers and incubated at 37 °C for 6 h to allow migration. Cell migration to the bottom of the filter was evaluated by measuring optical density at 590 nm. For Ki16425 treatment, cells were preincubated with 1 µM Ki16425 for 30 min.

Wound Healing Assay—Cells were plated in cell culture plates (12-well) using cell growth media containing fetal bovine serum. After the cells had reached semiconfluence, fetal bovine serum was removed from the media and replaced with serum-free media. A plastic pipette tip was drawn across the center of the plate to produce a clean wound area 24 h after serum depletion. Medium was then replaced with serum-free medium containing different concentrations of 1-oleoyl-LPA, 1-oleoyl-LPC, Ki16425 (1 µM) and lysoPLD. After the cells were cultured for 12, 24, or 36 h, cell movement into the wound area was examined. The migration distances between the leading edge of the migrating cells and the edge of the wound were compared.


Figure 1
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  		FIGURE 1.
Expression profiles of ATX and LPA receptorsintumorcelllines.AquantitativeRT-PCR analysis of ATX (left) and LPA receptors (LPA1, LPA2, LPA3, and LPA4, right) in 50 tumor cell lines from various origins (CNS, lung, breast, stomach, colon, kidney, ovary, prostate, cervix, fibroblast, and melanoma) was performed. Values are expressed relative to the expression of GAPDH mRNA.

 
Quantification of LPC—LPC concentration in cell culture supernatant was determined as described previously (35). Briefly, cells were cultured in serum-free RPMI 1640 containing 0.1% fatty acid-free bovine serum albumin (Sigma) for 2 days. LPC was extracted from culture media using the Bligh and Dyer method (51) and resuspended in phosphate-buffered saline containing 0.1% bovine serum albumin. LPC concentration was determined by a recently developed enzymatic colorimetric method as described. Briefly, samples were treated with lysophospholipase, glycerophosphorylcholine, phosphodiesterase, and choline oxidase. The resulting hydrogen peroxide generated was quantified using horseradish peroxidase and TOOS reagent.


Figure 2
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  		FIGURE 2.
ATX protein and lysoPLD activity in glioblastoma cell lines. A, Western blot of cell membrane fractions (ppt), soluble fraction (sup), and cell culture medium using anti-human ATX monoclonal antibody 3D1. B, lysoPLD activity of culture media from glioblastoma and other cancer cell lines. LPC was used as the substrate.

 
RNA Interference—SNB-78 glioblastoma cells were transfected with siRNA oligonucleotide duplexes 1 day after confluence (day -1) with trans-it TKO (Takara, Kyoto, Japan) according to the manufacturer's instructions. Generally 20 nM siRNA was transfected with 0.5 µl of Lipofectamine per well of a 24-well plate with fresh media. Each experiment contained equivalent samples transfected with a nontargeting control siRNA pool and samples not treated with trans-it TKO. siRNA oligonucleotide duplexes for each gene of interest were purchased from WAKO (Osaka, Japan) as optimized single duplexes (ATX1, sense, 5'-gccguuggagucaauaucuGC-3', antisense, 5'-agauauugacuccaacggcAA-3' and ATX2, sense, 5'-gggagacugcuguaccaauTA-3', antisense, 5'-auugguacagcagucucccCT-3'). Transfection efficiency was monitored using fluorescent (Cy3)-tagged oligonucleotides (Blockit, Invitrogen) transfected as described above and visualized with a mercury lamp fluorescent microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of ATX and LPA Receptors in 50 Tumor Cell Lines—We examined the expression of ATX in 50 cultured human tumor cell lines derived from various tumors using the quantitative RT-PCR technique. We found that some cells expressed a significant amount of ATX at both the mRNA and the protein levels (Figs. 1 and 2). High ATX expression was detected in DMS273 (lung cancer), colo320 (colon cancer), SKOV3 (ovarian cancer), MKN1 (stomach cancer), and most of the brain cancer cells (SF295, SF539, SF268, SNB-75, and SNB-78). The expression was highest in SNB-78 cells. In good agreement with this observation, both ATX protein and lysophospholipase D activity were detected in the culture supernatants of these ATX-positive cells (Fig. 2). Most of the protein was detected in the culture cell supernatants, whereas a small amount was detected in cells (Fig. 2A). These results confirm that ATX is secreted from cells, although ATX is initially biosynthesized in cells as a type II membrane protein.


Figure 3
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  		FIGURE 3.
Enhanced expression of ATX in GBM tissues. Expressions of ATX (A) and LPA receptors (LPA1, LPA2, LPA3, and LPA4) (B) in various brain tumor tissues as measured by quantitative RT-PCR. Of the three LPA receptors (LPAR), LPA1 has the highest expression in various brain tumors, possibly reflecting the high LPA1 expression in normal human brain. Note that tissues AS2 number 4 and GBM number 1 are derived from the same patient. The patient's cancer was initially diagnosed as astrocytoma, and after the recurrence, it was diagnosed as GBM.

 
We also examined the expression of the LPA receptors (LPA1, LPA2, LPA3, and LPA4) in the 50 human tumor cell lines using quantitative RT-PCR. Although the expression pattern of the four LPA receptors does not necessarily reflect the tissue origin of the tumor cells, restricted LPA receptor expression patterns were obtained (Fig. 1). LPA2 was predominantly expressed in cells from colon, stomach, and breast cancers (36, 37). LPA3 expression was relatively low. However, LPA3 was expressed by certain ovarian and prostate cancer cell lines. Expression of LPA4 was fairly low. By contrast, LPA1 was dominant in brain tumor cells (Fig. 2).


Figure 4
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  		FIGURE 4.
Overexpression of ATX in combination with LPC induces a migratory response in glioma cells in Boyden chamber assay. A, expression profiles of LPA receptors (LPA1, LPA2, LPA3, and LPA4) and ATX in mouse glioma cell line 203g as measured by quantitative RT-PCR. B, expression of ATX in stably transfected 203g-ATX cells as measured by Western blotting. mock, mock-transfected; MW, molecular mass markers. C, migratory responses of 203g-ATX (closed circles) and mock-transfected cells (open circles) to LPA and the effect of LPA1 antagonist, Ki16425, on the migratory response (closed squares). D, migratory responses of 203g-ATX cells (closed circles) and mock-transfected cells (open circles) to LPC and the effect of Ki16425 on the migratory response (closed squares).

 
Expression of ATX and LPA Receptors in GBM—Because SF295, SF539, SF268, SNB-75, and SNB-78 are defined as glioblastomas (gliomas derived from GBM), we attempted to examine the expression of ATX and LPA receptors in tissues from various brain tumors. We found that expression of ATX was markedly high in GBM tissues (Fig. 3). Three of four GBM tissue samples showed extremely high ATX expression. ATX expression is apparently lower in tissues from other brain tumors. One exception is a patient of astrocytoma (case AS2 number 4) whose tissue showed high ATX expression. He experienced early recurrence after only 16 months, and the tumor progressed to GBM at recurrence (case GBM number 1). Among the four LPA receptors, LPA1 was dominantly expressed in most brain tumor tissues tested including GBM with low expression of LPA2, LPA3, and LPA4, which may reflect the expression pattern in normal brain tissues (38, 39). The expression pattern of ATX and LPA receptors in GBM tissues indicates that ATX contributes to the invasive property of glioblastomas by producing LPA.

LPC Stimulates Cell Motility of ATX-expressing Cells—To test the possibility that glioblastomas acquire their high invasiveness through autocrine production of LPA by ATX, we first examined the effect of enhanced ATX expression on cell motility. We used mouse glioma cell line 203G that expressed LPA1 (Fig. 4A) but not a detectable amount of ATX (Fig. 4B). 203G glioma cells that stably express ATX (203G-ATX) were established by transfecting ATX cDNA and by selecting neomycin-resistant clones. The established three lines expressed significant levels of ATX as judged by both lysoPLD activity (data not shown) and Western blotting (Fig. 4B). In addition, these cell lines showed similar expression pattern of LPA receptors to the parental 203G cells (data not shown). The effects of LPA on the motility of transfected cells and mock-transfected 203G cells in the Boyden chamber were similar (Fig. 4C). LPA had a similar effect on the motility of parental 203G cells (not shown). The effect of LPA on the motility of these cells was abolished by the LPA1 antagonist, Ki16425 (Fig. 4C). By contrast, these cells showed quite distinct responses to LPC. LPC significantly stimulated the migration of 203G-ATX cells in Boyden chamber assay (Fig. 4D). However, a similar response was not induced in mock-transfected 203G cells (Fig. 4D) or in parental 203G cells (not shown). In addition, the stimulatory effect of LPC in 203G-ATX cells was completely abolished by the addition of Ki16425 (Fig. 4D), showing that LPA mediates the LPC-stimulated cell migration of the cells through LPA1. We confirmed that platelet-derived growth factor induced similar migratory response in ATX-overexpressing, mock-transfected, and parental 203G cells (data not shown).

We further examined the role of endogenously expressed ATX in the cell motility of ATX-expressing cells, which has not been previously demonstrated so far. For this experiment, we used SNB-78, which has the highest ATX expression among the 50 tumor cell lines (Fig. 1). As shown in Fig. 5A, SNB-78 cells, like other LPA1-positive cells, showed a migratory response to exogenous LPA in the Boyden chamber. LPC also stimulated the migration of SNB-78 (Fig. 5B). Ki16425 blocked not only the LPA-induced migratory response but also the LPC-induced migratory response (Fig. 5). This indicates that: 1) LPC is converted to LPA by the lysoPLD activity of endogenous ATX, 2) the LPA generated subsequently stimulates cell migration through LPA1, and 3) LPC behaves as a chemotactic factor toward ATX-expressing cells.


Figure 5
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  		FIGURE 5.
Effects of LPA and LPC on migration of glioblastoma cells. Migration of SNB-78 glioblastoma cells in response to LPA (A, closed circles) and LPC (B, closed circles) was measured with a Boyden chamber. Both responses were abolished by Ki16425 (open circles).

 
We previously showed that LPC is released from cells and could be converted to LPA to induce cell migration by exogenously added ATX (14). In fact, SNB-78 was found to release a small amount of LPC into the cell culture medium, and the amount gradually increased during the culture (data not shown). However, we were unable to evaluate the effect of cell-derived LPC on cell motility in the Boyden chamber since LPC is lost during preparation of the cells for the Boyden chamber assay. Accordingly, to evaluate the effects of cell-derived LPC and ATX on cell migration, we used a wound healing assay, which takes a relatively long period. As expected, exogenously added LPA was able to induce migration of SNB-78 cells in the wound healing assay (Fig. 6A). The migratory response was abolished by Ki16425, which shows that LPA1 is involved in this system, as was observed in Boyden chamber assay (Fig. 6A). In the absence of exogenous LPC, we observed a weak migratory response, which was also blocked by Ki16425 (Fig. 6B), showing that endogenous LPC and ATX, to a lesser extent, contribute to the migration of SNB-78 cells. By contrast, strong migratory responses were observed when LPC (10 µM) was added to the cells. The migratory response induced by LPC was again inhibited by Ki16425 (Fig. 6C). The amount of LPC released from SNB-78 was ~50 nM as judged by the enzymatic colorimetric method for determination of LPC. This indicates that the amount of endogenous LPC released from SNB-78 is insufficient to induce a full migratory response in the cells and that exogenous LPC is potentially a key factor in controlling the migratory response of ATX-expressing cells.


Figure 6
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  		FIGURE 6.
Glioblastoma migrates in response to LPA and LPC in wound healing assay. A clean wound area was produced on a monolayer of semiconfluent SNB-78 cells, seeded in 12-well plates. The wound was then allowed to heal for 24 h in serum-free media in the presence of LPA (10 µM, A) or LPC (10 µM, C) or in the absence of exogenous lipids (B). Cell morphologies before (0 h) and after (24 h) the treatment are shown in the upper and lower panels, respectively. The effect of Ki16425 (1 µM) on the cell migration is also shown in the right panels.

 


Figure 7
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  		FIGURE 7.
Down-regulation of ATX in SNB-78 cells using siRNA. One-day postconfluent SNB-78 cells were transfected with ATX (ATX1 and ATX2) or control siRNA duplexes. Forty-eight hours later, down-regulation of ATX in the cell supernatants was analyzed by measuring lysoPLD activity in the cell culture supernatant of the cells.

 


Figure 8
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  		FIGURE 8.
ATX produced by SNB-78 cells contributes to the motility of the cells. SNB-78 cells were transfected with ATX or control siRNA duplex to suppress ATX level, and after 24 h, the cells were subjected to wound healing assay both in the presence and in the absence of LPC (10 µM) for 24 h. The cells were also treated with LPA (10 µM) as a positive control. Cell morphologies before (0 h) and after (24 h) the treatment are shown in the upper and lower panels, respectively.

 
ATX Is Responsible for Motility of SNB-78 Cells in Wound Healing Assay—Our data suggest that endogenously expressed ATX in SNB-78 cells plays a key role in the motility of the cells by producing LPA from endogenously or exogenously supplied LPC. To confirm this hypothesis, we used siRNA to down-regulate the expression of ATX in SNB-78 cells. Confluent SNB-78 cells were transfected with varying amounts of siRNA duplexes using scramble siRNA as a control. As shown in Fig. 7, after 48 h of culture, successful down-regulation of ATX in the cell culture supernatants was confirmed by measuring lysoPLD activity of the culture cell supernatant. We next performed the wound healing assay using the ATX-down-regulated cells. We found that motility of SNB-78 cells was significantly suppressed both in the absence and in the presence of LPC, when the cells were treated with siRNA to suppress ATX expression (Fig. 8). Exogenously added LPA was able to induce migration of ATX-down-regulated cells (Fig. 8), showing that the siRNA-treated cells still retained the migratory activity in response to LPA. Thus, we concluded that ATX contributes to the cell migration of SNB-78 cells in the wound healing assay.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
GBM is the most malignant brain tumor due to its high invasiveness. In this study, we found that ATX, a cell motility-stimulating factor (4), is overexpressed in GBM tissues and many glioblastoma cell lines. ATX has catalytic activity to produce a potent chemoattractant-like lipid, LPA, and stimulates cell motility through an LPA G-protein-coupled receptor, LPA1 (14, 21). Interestingly, both GBM tissues and glioblastomas express high levels of LPA1, and in fact, the glioblastoma, SNB-78, used in this study showed migratory responses not only to LPA but also to LPC (Figs. 5 and 6). In addition, suppression of ATX expression in SNB-78 cells by siRNA resulted in dramatic reduction of migratory response of the cells to LPC but not to LPA (Fig. 8). Furthermore, LPA1 antagonist, Ki16425, effectively suppressed both LPA- and LPC-induced motility of glioblastoma cells. Thus, the motility of glioblastoma cells appears to depend on ATX and LPA1. LPA-induced cell motility of glioblastomas was also shown in the previous report by Manning et al. (16).

Enhanced ATX expression has been repeatedly demonstrated in various tumors, including non-small cell lung cancer, breast cancer, renal cell cancer, hepatocellular carcinoma, and thyroid cancer (6-12). In breast cancer, ATX expression level strongly correlates with the invasiveness of cancer cells (8). Thus, it is reasonable to assume that ATX expressed by tumor cells is responsible for the motility, and thus, the invasiveness of the cells. However, the spontaneous motility of non-small cell lung cancer cells in vitro did not correlate with the levels of ATX mRNA (6), indicating that other factors may influence ATX-induced cell motility. In this study, we used glioblastoma cell lines that endogenously express ATX and cell transformants overexpressing ATX and evaluated ATX, LPA1, and LPC, a substrate for ATX of lysoPLD activity. Our results show that LPC is a critical factor that regulates ATX-mediated cell motility. We previously showed that LPC is synthesized and released from various tumor cells and that it is a potential substrate for ATX when ATX is added to the cells in the absence of exogenous LPC (14). However, the concentration of cell-derived LPC (~50 nM) is too low to induce full cell motility of SNB-78 cells (Fig. 6). Although a high dose of exogenously added ATX induced cell motility (by converting cell-derived LPC to LPA), the amounts of endogenously expressed ATX in glioblastoma cells and even in ATX-overexpressing glioma cells were insufficient to have an effect on cell motility. We found that the addition of exogenous LPC to the ATX-expressing cells but not ATX-negative cells strongly induced cell motility, although LPA equally promoted the motility of both cell types (Figs. 4, 5, 6). Thus, LPC is a chemotactic factor for tumor cells expressing ATX and LPA1. LPC has previously been shown to act as a chemotactic factor for other cell types, including macrophages (40), monocytes (41), and T-lymphocytes (42). In some cell types, such as macrophages, cell motility appears to be induced by another system (LPC-specific G-protein-coupled receptor, G2A) (43), whereas in other cell types, cell motility appears to be induced by the ATX-LPA1 system.

In general, the BBB is disrupted in GBM tissue, which leads to exposure of tumor cells to components of plasma that might affect the cell motility of glioblastomas (31, 32). As indicated by Manning et al. (16), one such plasma component may be LPA. However, LPA concentration in plasma is quite low (below 50 nM), although in serum, it may be above 1 µM (44, 45). In contrast, LPC concentration in plasma is extremely high (100-300 µM in human and ~500 µM in rats) (35, 44). The concentration of LPC in brain tissue, such as in cerebrospinal fluids, is quite low (several µM level) (46). Thus, when the BBB is disrupted in GBM, it is likely that these tumors are exposed to LPC derived from plasma. LPC is converted to LPA by ATX expressed by glioblastomas, and consequently, LPA1 is activated, leading to the enhanced cell motility and high invasiveness of GBM (Fig. 9). This idea is supported by the finding that GBM cells travel along blood vessels (47).


Figure 9
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  		FIGURE 9.
A schematic model showing the effects of ATX, LPA1, LPA, and LPC on the motility of GBM cells upon the disruption of BBB. On disruption of BBB, GBM in CNS parenchyma is likely to be exposed to LPC derived from plasma. Then LPC is converted to LPA by ATX expressed by GBM and subsequently activation of LPA1, leading to the enhanced motility and high invasiveness of GBM. EC, endothelial cells; BM, basement membrane.

 
The expression patterns of LPA receptors in the 50 cancer cell lines (Fig. 1) agree with the previous reports on LPA receptor expression. For example, LPA3 expression is relatively high in cells from ovarian cancer (48) and, markedly, LPA2 is predominantly expressed in cells derived from colon (37) and thyroid cancer (49). Recently, we also observed similar LPA receptor distribution patterns in human breast cancers (36) in which LPA2 expression is dramatically enhanced. The present study revealed that LPA1 is predominantly expressed in GBM cells and tissues. The same is true for ATX. Thus, the expression patterns in cancer cell lines are definitely informative to understand the roles of ATX and LPA receptors in cancer cell biology.

ATX may contribute to cancer cell survival and motility in several ways. In addition to its cell motility-stimulating activity, ATX has a cell proliferation-stimulating activity (14) and an angiogenic factor-like activity that induces new vessel formation by an unknown mechanism (50). GBM is known to induce vascular proliferation, and so ATX might have a role here as well. In summary, our results show that ATX, a potent oncogenic protein, is overexpressed in GBM and stimulates the motility of glioblastomas. Thus, ATX is a potential diagnostic marker for GBM. In addition, ATX, LPA1 and unidentified LPC-producing enzymes are potential targets for GBM therapy.


   FOOTNOTES
 
* 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. Back

1 To whom correspondence should be addressed. Tel.: 81-3-5841-4723; Fax: 81-3-3818-3173; E-mail: jaoki{at}mol.f.u-tokyo.ac.jp.

2 The abbreviations used are: ATX, autotaxin; GBM, glioblastoma multiforme; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; lysoPLD, lysophospholipase D; RT, reverse transcription; BBB, blood-brain barrier; NPP, nucleotide pyrophosphatases/phosphodiesterases; CNS, central nervous system; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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