Received for publication, March 23, 2001, and in revised form, April 23, 2001
Elevated tumor cyclooxygenase (COX-2) expression
is associated with increased angiogenesis, tumor invasion, and
suppression of host immunity. We have previously shown that genetic
inhibition of tumor COX-2 expression reverses the immunosuppression
induced by non-small cell lung cancer (NSCLC). To assess the impact of COX-2 expression in lung cancer invasiveness, NSCLC cell lines were
transduced with a retroviral vector expressing the human COX-2 cDNA
in the sense (COX-2-S) and antisense (COX-2-AS) orientations. COX-2-S
clones expressed significantly more COX-2 protein, produced 10-fold
more prostaglandin E2, and demonstrated an
enhanced invasive capacity compared with control vector-transduced or
parental cells. CD44, the cell surface receptor for hyaluronate, was
overexpressed in COX-2-S cells, and specific blockade of CD44
significantly decreased tumor cell invasion. In contrast, COX-2-AS
clones had a very limited capacity for invasion and showed diminished
expression of CD44. These findings suggest that a COX-2-mediated,
CD44-dependent pathway is operative in NSCLC invasion.
Because tumor COX-2 expression appears to have a multifaceted role in
conferring the malignant phenotype, COX-2 may be an important target
for gene or pharmacologic therapy in NSCLC.
 |
INTRODUCTION |
Cyclooxygenase (also referred to as prostaglandin endoperoxidase
or prostaglandin G/H synthase) is the rate-limiting enzyme for the
production of prostaglandins
(PGs)1 and thromboxanes from
free arachidonic acid (1). The enzyme is bifunctional, with fatty acid
cyclooxygenase (producing PGG2 from arachidonic acid) and
PG hydroperoxidase activities (converting PGG2 to
PGH2). Two forms of cyclooxygenase (COX) have now been described: a constitutively expressed enzyme, COX-1, present in most
cells and tissues, and an inducible isoenzyme, COX-2 (also referred to
as PGS-2), expressed in response to cytokines, growth factors, and
other stimuli (1-4). COX-2 has been reported to be constitutively
overexpressed in a variety of malignancies (4-11); we and others have
reported that COX-2 is frequently constitutively elevated in human
NSCLC (12-16). Previous studies indicate that overexpression of tumor
COX-2 may be important in tumor invasion (17, 18), angiogenesis (19,
20), resistance to apoptosis (21-23), and suppression of host immunity
(13, 24). Our current studies focus on the role of tumor COX-2
expression in modulating NSCLC invasion.
Tumor metastasis is a complex series of events in which cells migrate
beyond tissue compartments and spread to distant organ sites. Cell
surface CD44, the receptor for hyaluronate, has an important
role in regulating tumor growth and metastasis because it mediates
cellular adhesion to extracellular matrix, which is prerequisite for
tumor cell migration (25, 26).
While COX-2 expression has previously been linked to enhanced matrix
metalloproteinase (MMP) expression and invasion (27), the role of CD44
in this COX-2-induced invasion has not been defined. Here, we report
that stable overexpression of COX-2 in NSCLC results in up-regulation
of CD44. Furthermore, we demonstrate a CD44-dependent increase in invasion in Matrigel matrix assays. In contrast,
abrogation of tumor-COX-2 expression results in decreased
PGE2 production, diminished CD44 expression and decreased
invasion. This is the first report documenting the critical role of
tumor COX-2 expression in the regulation of CD44-dependent
invasion by human NSCLC.
 |
EXPERIMENTAL PROCEDURES |
Transfection Protocol--
A 2.0-kilobase pair cDNA
fragment of human COX-2 (generously provided by Dr. Harvey Herschman,
UCLA) was cloned into the PmeI site of the retroviral vector
pLNCX (CLONTECH, Palo Alto, CA). In this vector,
transcription of the COX-2 cDNA is controlled by the
cytomegalovirus promoter, and transduced cells can be enriched under G418 selection. Sense (COX-2-S) and antisense (COX-2-AS) oriented
expression vectors were prepared. A549 (human lung adenocarcinoma) and
H157 (squamous cell carcinoma) cells were obtained from American Type
Culture Collection (ATCC, Manassas, VA) and the National Cancer
Institute, respectively. Tumor cells were transfected with COX-2-S,
COX-2-AS-expressing vectors, or pLNCX (vector alone) using
calcium-phosphate-mediated transfection (Promega, Madison, WI). The
transfected cells were grown in an atmosphere of 5% CO2 in
air at 37 °C in cell culture medium consisting of RPMI 1640 (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (Gemini Biological Products, Calabasas, CA), 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM glutamine (Life
Technologies, Inc.). For selection, the medium also contained 0.5 mg/ml active G418 (Life Technologies, Inc.). Following G418
selection, the polymerase chain reaction (PCR) was used to confirm the
positive COX-2-S and COX-2-AS clones. The PCR was done using the pLNCX 5' primer (AGCTTCGTTTAGTGAACCTCAGATCG) and pLNCX 3' primer
(ACCTACAGGTGGGGTCTTTCATTCCC). PCR positive clones were then screened by
Western blot and EIA analysis for COX-2 expression and PGE2
production, respectively. For each tumor cell line a high
COX-2-expressing and PGE2-producing clone for COX-2-S, and
a low COX-2-expressing and PGE2-producing COX-2-AS clone,
were identified from a survey of 25 clones. These clones were then
expanded for further studies.
Measurement of Prostaglandin E2--
Control,
COX-2-S, and COX-2-AS cells (A549 and H157) were stimulated with
IL-1
(200 units/ml, Genzyme, Cambridge, MA) for 24 h.
PGE2 concentration in each group (with or without IL-1
stimulation) were measured by EIA using a PGE2 EIA kit
(Cayman Chemical, Ann Arbor, MI) as reported previously (28). All
measurements were made in triplicate and repeated in at least three
separate experiments.
Western Blot Analysis for COX-2 and CD44 Expression--
The
cells from each treatment group were lysed at 4 °C for 15 min in
lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl,
1.5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.6% Nonidet P-40). The
cell lysates were centrifuged at 13,000 rpm for 10 min and the
supernatant collected. Total protein was measured with a protein assay
reagent (Bio-Rad), and 20 µg of cell lysate protein were
separated on 10% SDS-polyacrylamide gels. Following separation, the
proteins were transferred to Hybond nitrocellulose membranes (Amersham
Pharmacia Biotech) and the filters probed with anti-human COX-2
antibody (Cayman Chemical). For CD44 detection, the filters were probed
with anti-human CD44 antibody 4A4 (29). The membranes were developed by
the ECL chemiluminescence system (Amersham Pharmacia Biotech) and
exposed to x-ray film (Fujifilm, Fuji Medical Systems Inc.,
Stamford, CT). Equal loading of samples was confirmed by probing the
membranes with
-actin antibody.
Invasion Assay--
To quantify invasion, the membrane invasion
assay was carried out in Matrigel-coated invasion chambers
(Becton Dickinson Labware, Franklin Lakes, NJ). Control and COX-2
transfectants were cultured in RPMI 1640 supplemented with 10% fetal
bovine serum. Tumor cells in log phase growth were detached by
trypsin-EDTA (Mediatech) and resuspended in RPMI 1640 with 0.1% bovine
serum albumin. Serum-free A549- and H157-conditioned medium was
obtained by incubation of these cells for 24 h. This tumor
cell-conditioned medium was added in the lower chamber as a
chemoattractant, and the resuspended cells (5 × 104)
were plated in the upper chamber. Following 18-h incubation at 37 °C
in a humidified 5% CO2 atmosphere, the cells in the upper chamber and on the Matrigel were mechanically removed with a
cotton swab. The cells adherent to the outer surface of the membrane were fixed with methanol and stained with hematoxylin/eosin. The invading cells were examined, counted, and photographed by microscopy (Nikon Labphot-2 Microscope with an attached Spot Digital Camera, A. G. Heinz, Lake Forest, CA) at × 50 magnification. Six fields were counted per filter in each group, and the experiment was repeated
five separate times. To assess the role of CD44 in mediating Matrigel invasion, the cells from COX-2-S-expressing clones were plated in the presence of 500 ng of anti-CD44 antibody (The Binding Site, Inc., San Diego, CA) or control mouse IgG (Dako Corp.,
Carpenteria, CA) and the cell numbers determined as described.
 |
RESULTS |
Expression of COX-2 and PGE2 Production in
COX-2-transduced NSCLC Cells--
To evaluate the role of COX-2
expression in mediating the lung cancer invasiveness, two NSCLC cell
lines were stably transduced with a retroviral vector encoding COX-2
and selected for G418 resistance. The COX-2-S clones (A549-S and
H157-S) exhibited enhanced constitutive COX-2 protein expression by
immunoblot (Fig. 1, A and
B). In contrast, COX-2 was not detectable in COX-2-AS clones (Fig. 1, A and B). The COX-2 protein expression
in the sense clones was found to be significantly higher (3-fold for
H157 and 10-fold for A549 cells), as measured by densitometry, than
that of parental or vector-transduced cells (data not shown). Compared
with parental and vector-transduced cells, COX-2-S clones (H157-S and
A549-S) exhibited a 5-12-fold increase in PGE2 production.
In contrast, COX-2-AS clones showed decreased constitutive
PGE2 production (Fig. 2,
A and B). IL-1
is one of several cytokines
known to potently up-regulate COX-2 expression in a variety of cells
(30). Consistent with our previous findings (28), parental and
vector-transduced control cells exhibited a 5-10-fold increase in
PGE2 production in response to IL-1
(Fig. 2,
A and B). In contrast, a similar induction of
PGE2 secretion was not observed in COX-2-AS clones in the
presence of IL-1
(Fig. 2, A and B).

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Fig. 1.
Elevated COX-2 expression in COX-2 modified
NSCLC cells. NSCLC cells (parental (P), empty vector
(V), COX-2-S (S), and COX-2-AS (AS)
transfectants) were cultured in the presence or absence of IL-1 (280 units/ml) for 24 h. Following incubation, 20 µg of cell lysate
was fractionated on a 10% SDS-PAGE gel and transferred to
nitrocellulose membranes and probed with COX-2-specific antibody A,
A549 cells. COX-2-S (S) exhibits enhanced constitutive COX-2
protein, while COX-2-AS shows decreased protein production compared
with controls (parental and vector). Increase in COX-2 expression in
response to IL-1 was only observed in control (parental and vector)
but not in COX-2-S or COX-2-AS cells. B, H157 cells. COX-2-S
cells exhibit COX-2 protein expression pattern that is similar to the
A549 cells. Equal loading of cell lysate was confirmed with
anti- -actin antibody.
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Fig. 2.
Increased PGE2 production in
COX-2 modified NSCLC cells. NSCLC cells (106 cells/ml
of parental (P), empty vector (V), COX-2-S
(S), and COX-2-AS (AS) transfectants) were
cultured in complete medium with and without IL-1 (280 units/ml).
Following a 24-h incubation the supernatants were collected for
PGE2 determination by enzyme immunoassay. A,
A549 cells. Compared with parental and vector controls a 10-fold
increase in constitutive PGE2 production is seen in COX-2-S
cells (p < 0.01), while an inhibition of
PGE2 production is seen in COX-2-AS cells
(p < 0.05). B, H157 cells. Compared with
parental and vector a 5-fold increase in constitutive PGE2
production is seen in COX-2-S cells (p < 0.05), while
an inhibition of PGE2 production is seen in COX-2-AS cells
(p < 0.05). In response to IL-1 , increase in
PGE2 production is seen in parental and vector control of
both cell lines. The PGE2 EIA results are expressed as
ng/ml/106 cells/24 h and are representative of four
experiments performed in duplicate. Bars, S.E.
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Overexpression of COX-2 Enhances the Invasive Capacity of NSCLC
Cells--
COX-2 overexpression may be associated with enhanced
invasiveness and metastasis in a variety of malignancies (27, 31). To
examine its role in NSCLC, we tested cells overexpressing COX-2 in
Matrigel matrix invasion assays. As depicted in Fig.
3A, COX-2-S cells were
significantly more invasive than parental or antisense-transfected A549
and H157 cells. The invasiveness was found to be similar for both of
the COX-2-S cell lines. By counting cells in random microscopic fields,
we determined that the sense clones of both A549 and H157 were 3-fold
more invasive than parental or vector-transfected controls (Fig.
3B). Furthermore, consistent with their undetectable COX-2
expression, COX-2-AS cells demonstrated a decreased invasiveness through Matrigel (Fig. 3, A and B).

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Fig. 3.
COX-2-S-modified NSCLC cells demonstrate
enhanced invasive capacity. NSCLC cells (parental
(P), empty vector (V), COX-2-S (S),
and COX-2-AS (AS) transfectants) were used for
Matrigel matrix assay. A, left panel,
A549; right panel, H157. Significantly more COX-2-S cells
invaded the matrix, while COX-2-AS cells showed decreased invasion
compared with parental and vector. Anti-CD44 monoclonal antibody, but
not control antibody, blocked the enhanced invasion of COX-2-S cells in
both cell lines. B and C, bar graphs
representing the number of cells that invaded in each group. A 3-fold
increase in number of cells invading the matrix is seen in COX-2-S
cells: B, A549 (p < 0.05) and C,
H157 (p < 0.05).
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NSCLC COX-2-dependent Invasion Is Mediated through CD44
Expression--
Because tumor CD44 expression is known to play an
important role in tumor invasion and metastasis (26, 32), we
hypothesized that CD44 expression may be important for NSCLC
COX-2-dependent invasion. Indeed, CD44 expression was
augmented in COX-2-S clones as determined by Western blot analysis
(Fig. 4, A and B).
In contrast, CD44 was not detectable in COX-2-AS cells (Fig. 4,
A and B). Consistent with the expression of COX-2
and PGE2 production, CD44 expression was also found to be
up-regulated in parental tumor cells in the presence of IL-1
. This
suggested that related signaling events could be mediating increased
COX-2 and CD44 expression in NSCLC. Because IL-1
could not induce
COX-2 activity or CD44 expression in the COX-2-AS transfectants, COX-2
appears to be a proximal regulator of CD44 expression in NSCLC.

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Fig. 4.
CD44 expression is up-regulated in
COX-2-S-modified NSCLC cells. The cell lysates from NSCLC cells
(parental (P), empty vector (V), COX-2-S
(S), and COX-2-AS (AS) transfectants) in the
presence or absence of IL-1 were produced, and 20 µg of lysate was
fractionated on a 10% SDS-PAGE gel. CD44-specific monoclonal antibody
was used to probe the membranes. An increase in CD44 protein is seen in
COX-2-S, while a decrease in CD44 protein is seen in COX-2-AS.
A, A549 cells. In response to IL-1 , significant increase
in CD44 protein was seen in parental, vector, and COX-2-S cells.
B, H157 cells. Increase in CD44 protein was observed in
parental, vector, and COX-2-S cells but not in COX-2-AS cells. Equal
loading of cell lysate was confirmed with anti- -actin
antibody.
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Consistent with the findings of Western blot analysis, FACS analysis of
the COX-2 transfected cells with a fluorescein
isothiocyanate-labeled CD44 antibody showed an increase in the
expression of CD44 in COX-2-S but decreased expression in COX-2-AS
cells (data not shown). To determine the importance of CD44 expression
in tumor COX-2-dependent invasion, we performed antibody
blocking experiments with anti-CD44 antibody. The enhanced invasiveness
of the COX-2-S clones was found to be inhibited with anti-CD44
antibody, whereas control monoclonal antibody had no effect (Fig. 3,
A and B). Anti-CD44 antibody did not completely
decrease COX-2-S invasion to the level of COX-2-AS cells, suggesting
that CD44-independent mechanisms may also be operative.
 |
DISCUSSION |
Mounting evidence from several studies indicates that tumor COX-2
activity has a multifaceted role in conferring the malignant and
metastatic phenotypes. The data in the current study implicates COX-2
overexpression as a proximal mediator of CD44-dependent invasion in human NSCLC. We demonstrate that augmenting COX-2 expression leads to increased invasion by NSCLC cells. Importantly, this enhanced invasion is associated with increased CD44 expression, and blocking CD44 abrogates the COX-2-mediated enhancement of NSCLC
cell mobilization through Matrigel.
Although multiple genetic alterations are necessary for lung cancer
invasion and metastasis, COX-2 may be a central element in
orchestrating this process (13-17). Studies indicate that
overexpression of COX-2 is associated with apoptosis resistance
(21-23, 33), angiogenesis (19, 20, 23, 34), decreased host immunity (13, 24), and enhanced invasion and metastasis (27). We reported
previously that COX-2 is overexpressed in human NSCLC and the resultant
high level PGE2 production-mediated dysregulation of host
immunity by altering the balance of interleukins 10 and 12 (13).
Indeed, specific inhibition of COX-2 led to significant in
vivo tumor reduction in murine lung cancer models (24). Recently, other studies have corroborated and expanded on our initial findings documenting the importance of COX-2 expression in lung cancer (12,
14-16, 35, 36). COX-2 activity can be detected throughout the
progression of a premalignant lesion to the metastatic phenotype (14).
Markedly higher COX-2 expression was observed in lung cancer lymph node
metastasis compared with primary adenocarcinoma (14). These reports,
together with studies documenting an increase in COX-2 expression in
precursor lesions (15, 16), suggest the involvement of COX-2
overexpression in the pathogenesis of lung cancer. Epidemiological
studies that indicate a decreased incidence of lung cancer in subjects
who regularly use aspirin have been interpreted as supporting this
hypothesis (37).
In addition to regulating immune responses, tumor COX-2 has been
implicated in inhibiting apoptosis (21, 38) and angiogenesis (3, 20).
The inhibition of programmed cell death in COX-2-expressing cells is
found to be associated with an increase in Bcl2 expression and a
decrease in the expression of both the TGF-
2 receptor and E-cadherin
protein (21). Experimental evidence suggests that ligation of the cell
surface matrix adhesion receptor CD44 by anti-CD44 antibody induces
cell detachment and triggers apoptosis in a variety of cells (39).
Thus, inhibiting anchorage dependence mediated by CD44 may contribute
to induction of apoptosis (40).
Overexpression of COX-2 also enhances tumor invasiveness and thus may
increase metastatic potential (3, 27). Tumor cell invasion involves the
active movement of cells across the extracellular matrix (41). Adhesion
to extracellular matrix, a critical initial step in the metastatic
process, has been found to be CD44-dependent in several
tumors (26, 32, 42, 43). CD44 is a receptor for hyaluronate, a major
glycosaminoglycan component of the extracellular matrix. In this
capacity, CD44 also serves to induce co-clustering with MMP-9 and can
therefore promote MMP-9 activity, tumor invasion, and angiogenesis (25,
44). Our findings indicate that tumor COX-2 overexpression in human
NSCLC constitutes an important driving force for CD44 induction. Thus,
COX-2 expression may form the basis for an important tumor-induced
invasive pathway. The fact that CD44-induced MMP-2 and -9 have the
capacity to activate latent TGF-
suggests an autocrine and paracrine
pathway in which collagen deposition and further invasion may be
enhanced (44, 45). The activation of latent TFG
also provides an
additional pathway for tumor-induced immune suppression (44, 46, 47).
In addition, recent studies by Sun et al. (48) demonstrate
that hyaluronate fragments have the capacity to up-regulate COX-2 by a
CD44-dependent pathway. Thus, hyaluronate itself may serve
to further enhance tumor COX-2 and CD44 expression leading to
maintenance of the COX-2-dependent invasive phenotype.
Lung cancer is the leading cause of cancer death in men and women in
the United States (49). Despite therapeutic efforts, 5-year survival in
lung cancer patients is less than 15% (50). Defining new molecular
targets will lead to more effective therapeutic strategies. Here we
document for the first time a pathway whereby COX-2 overexpression
leads to CD44-dependent invasion in NSCLC. These findings
suggest that therapies targeting COX-2 may diminish the propensity for
invasion and metastases in NSCLC.
The abbreviations used are:
PG, prostaglandin;
COX, cyclooxygenase;
NSCLC, non-small cell lung cancer;
MMP, matrix metalloproteinase;
PCR, polymerase chain reaction;
EIA, enzyme immunoassay;
IL, interleukin;
TGF, transforming growth factor;
PAGE, polyacrylamide gel electrophoresis.
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