Cyclooxygenase-2 Inhibition Suppresses A v B 6 Integrin – Dependent Oral Squamous Carcinoma Invasion

Worldwide oral squamous cell carcinoma (OSCC) represents about 5.5% of all malignancies, with f30,000 new cases each year in the United States. The integrin AvB6 and the enzyme cyclooxygenase-2 (COX-2) are implicated in OSCC progression and have been suggested as possible therapeutic targets. Each protein also is reported to identify dysplasias at high risk of malignant transformation, and current clinical trials are testing the efficacy of nonsteroidal anti-inflammatory drugs (NSAID) at preventing OSCC development. Given the probable increased expression of AvB6 and COX-2 in OSCC and the inhibition of several integrins by NSAIDs, we investigated whether NSAIDs affected AvB6-dependent cell functions. We found that expression of both AvB6 and COX-2 was significantly higher in OSCC compared with oral epithelial dysplasias. Neither protein preferentially identified those dysplastic lesions that became malignant. Using OSCC cell lines, modified to express varying levels of AvB6, we assessed the effect of COX-2 inhibition on cell invasion. We found that the COX-2 inhibitor NS398 inhibited specifically AvB6-dependent, but not AvB6-independent, OSCC invasion in vitro and in vivo, and this effect was modulated through prostaglandin E2 (PGE2)–dependent activation of Rac-1. Transient expression of constitutively active Rac-1, or addition of the COX-2 metabolite PGE2, prevented the anti-invasive effect of NS398. Conversely, RNA interference down-regulation of Rac-1 inhibited AvB6-dependent invasion. These findings suggest that COX-2 and AvB6 interact in promoting OSCC invasion. This is a novel mechanism that, given the ubiquity of AvB6 expression by head and neck cancers, raises the possibility that NSAIDs could protect against OSCC invasion. (Cancer Res 2006; 66(22): 10833-42)


Introduction
Cyclooxygenases (COX) catalyze the key step in prostanoid and thromboxane biosynthesis and are targets of nonsteroidal antiinflammatory drugs (NSAID).Two human isoforms exist: COX-1, expressed constitutively in most mammalian cells, generates prostaglandins necessary for normal physiologic function, whereas COX-2, normally undetectable, is induced rapidly by stimuli, including cytokines, oncogenes, and tumor promoters (1,2).Elevated COX-2 expression occurs in many carcinomas, including oral squamous carcinoma (OSCC), where it contributes to tumor progression (1)(2)(3).Transgenic female mice overexpressing COX-2, for example, have a high frequency of breast dysplasia and metastatic tumors (4), whereas genetic inactivation of COX-2 in a murine model of familial adenomatous polyposis reduced both the size and number of intestinal polyps (5).
The epithelial integrin a v h 6 is a receptor for fibronectin, vitronectin, tenascin, and the latency-associated peptide (LAP) of transforming growth factor-h (e.g., ref. 6).Generally found to have minimal expression in adult epithelium, a v h 6 is up-regulated during epithelial remodeling (e.g., wound healing) and tumorigenesis.Aberrant a v h 6 expression occurs in numerous carcinoma types, particularly OSCC (7)(8)(9)(10)(11).Moreover, increased expression of both a v h 6 and COX-2 have been reported in oral dysplastic epithelium, suggesting a role in tumor progression (12,13).
NSAIDs have marked antitumor activity via COX-2 inhibition.Selective COX-2 inhibitors are chemopreventive in animal models of colon, bladder, and breast cancer (1,2), whereas epidemiologic studies indicate that NSAIDs reduce breast, esophageal, and colon cancer mortality (1,2).The effects of NSAIDs on tumor growth and progression are likely to be through multiple mechanisms, including indirect effects on angiogenesis and inflammation, as well as direct effects on tumor cell proliferation and apoptosis (1,2).Particularly interesting are studies where NSAIDs inhibit tumor cell adhesion, migration, and invasion (1,2,14).These changes may be mediated via effects on various integrins (15,16).For example, aspirin and indomethacin inhibit activation of the platelet integrin a IIb h 3 (17) whereas Dormond et al. inhibited endothelial cell migration and angiogenesis using the selective COX-2 inhibitor NS398, effects that were modulated through inhibition of the a v h 3dependent activation of Rac-1 and Cdc42 (18).This may be a reciprocal effect because integrin-mediated adhesion can contribute to stabilization of COX-2 protein levels (19).
Given that both a v h 6 and COX-2 are expressed in OSCC and have been reported to promote invasion (20)(21)(22), and that NSAIDs may inhibit integrin function, we determined whether NSAIDs could inhibit a v h 6 -mediated invasive activity.
Cell culture.Using cDNA transfection techniques, we created a panel of cell lines expressing various levels of a v h 6 (20,21).H357 is an a v -negative OSCC cell line (24) from which the V3 cell line was generated by transfection of a v cDNA (25); V3 cells express low levels of a v h 6 .This cell line was retrovirally infected with h 6 cDNA, creating the VB6 cell line, which has high a v h 6 expression.A null transfectant control cell line for the VB6 cells (C1) also was generated at this time (20,21).BICR6, CA1, and H157 OSCC cell lines were also used: provided by Professors K. Parkinson (BICR6) and I. Mackenzie (CA1) of the Queen Mary's University, London and Professor S. Prime (H357 and H157) of the University of Bristol Dental School.OSCC cells were grown in keratinocyte growth medium (KGM; ref. 20).Human foreskin fibroblasts (supplied by Cell Services of Cancer Research UK, London Research Institute) were maintained in fibroblast growth medium (DMEM supplemented with 10% FCS) at 37jC in an humidified atmosphere.
Flow cytometry.Flow cytometry was done (20), using anti-a v h 6 antibody (E7P6; Chemicon International, Harrow, United Kingdom) and FITC-conjugated secondary antibody (DAKO).Negative control used secondary antibody only and was subtracted from the results.Labeled cells were scanned on a FACSCalibur cytometer (BD Biosciences, Oxford, United Kingdom) and analyzed using CellQuest software, acquiring 1 Â 10 4 events.Results show mean fluorescence (arbitrary units, log scale).
RNA interference.RNA interference (RNAi) SMART pool reagents targeting h 6 , Rac-1, COX-2, or control (random) sequences were obtained from Dharmacon (Chicago, IL).Cells were seeded into six-well plates and left for 24 hours until f40% confluent, then transfected with 100 pmol/well of the relevant duplex pool using Oligofectamine transfection reagent (Invitrogen, Paisley, United Kingdom).Cells were used in assays after 24 to 48 hours.Cells were also lysed and used to verify protein knockdown by Western blotting analysis.
Organotypic culture.Organotypic cultures with an air-tissue interface were prepared as described (26).Gels comprised a 50:50 mixture of Matrigel (Becton Dickinson, Oxford, United Kingdom) and type I collagen (Upstate) containing 5 Â 10 5 /mL human foreskin fibroblasts, to which were added 5 Â 10 5 OSCC cells.For inhibition studies, NS398 (20 Amol/L) with or without PGE 2 (1-4 ng/mL) was added to the KGM.The medium was changed every 2 days.After 6 or 14 days, the gels were bisected, fixed in formal-saline, and processed to paraffin.Four-micrometer sections were immunostained with the pan-cytokeratin antibody AE1/AE3 (DAKO).

Cancer Research
Cancer Res 2006; 66: (22).November 15, 2006 Quantitative analysis.Invasion in organotypic culture was quantified as described (26).Immunostained sections were viewed at Â100 magnification and digitally photographed.The digital image was converted to greyscale (Optilab 2.6.1,Graftek Imaging, Inc., Austin, TX), and an ''invasion index'' was calculated from this image.This gave a quantitative value for tumor invasion (the product of the average depth of invasion, the number of invading particles, and the area of the invading particles; ref. 26).Three 4-Am sections spaced at 150 Am were analyzed for each in vitro gel.
Transplantation of organotypic cultures onto nude mice.All animal experiments were done according to the guidelines of the local ethics committee and were as specified in the project license.Organotypic cultures were grown in vitro for 7 days before being transplanted into 10-week-old athymic nude (nu/nu) mice as described previously (26).Seven days after transplant, test animals were given 5 mg/kg NS398 i.p. every 72 hours, whereas control animals received vehicle (1:100 DMSO in PBS) only.At 6 weeks, animals were killed, and the transplants were dissected en bloc and further processed for histology.Five 4-Am sections spaced at 150 Am were analyzed for each in vivo gel.Sections where the tumor epithelium deviated significantly from the horizontal were excluded from analysis.
Statistical analysis.Data are expressed as the mean F SD of a given number of observations.Where appropriate, one-way ANOVA was used to compare multiple groups.Comparisons between groups were by Fisher's PLSD.P < 0.05 was considered significant.Figures show representative examples of independent repeats with error bars representing SD unless stated otherwise.

Results
A v B 6 and COX-2 expression in hyperplastic and dysplastic epithelium and in OSCC.We examined the expression and distribution of a v h 6 and COX-2 expression in hyperplastic and dysplastic epithelium and OSCC by immunochemistry.Results are summarized in Table 1.Of 39 dysplastic lesions examined, moderate (++) to strong (+++) expression of a v h 6 and COX-2 was present in 21% and 31%, respectively.Neither protein preferentially identified lesions, which subsequently transformed to OSCC, and similar staining sometimes was found in hyperplastic epithelium.In contrast, a v h 6 and COX-2 were both up-regulated in established OSCC, with moderate to strong expression in 85% and 80% of tumors, respectively.Staining for both proteins often was more intense at the periphery of invading tumor islands (Fig. 1).
COX-2 inhibition suppresses A v B 6 -dependent cell invasion in Transwell assays.From the H357 a v -negative OSCC line, a high a v h 6 expressing line (VB6) was generated using cDNA transfection and retroviral infection (20,21).A null transfectant line (C1), expressing low levels of a v h 6 , also was created (Fig. 2B; ref. 20).As shown previously, VB6 cells are significantly more invasive than C1 control cells, and the increased invasion is a v h 6 dependent (Fig. 2A; ref. 20).
The cell lines were examined for COX-2 expression by Western blotting.All three lines expressed the 72-kDa COX-2 protein at similar levels (Supplementary Fig. S1A).An additional band (f65 kDa), corresponding in size with unglycosylated COX-2 (3), was apparent, possibly reflecting synthesis of new COX-2 protein.Inhibition of a v h 6 with blocking antibodies did not effect COX-2 expression in VB6 cells (data not shown).
We assessed the effects of NSAIDs on a v h 6 -dependent OSCC Transwell invasion using SC-560 (COX-1 inhibitor), NS398 (COX-2 inhibitor), and indomethacin (nonselective COX inhibitor).Over 16 experiments, invasion of the high a v h 6 -expressing VB6 cells was inhibited significantly using NS398 (Fig. 2A; P < 0.00001).Indomethacin produced similar results (Fig. 2A; P < 0.0001).COX-2 inhibition in VB6 cells reduced invasion to control C1 levels, comparable with inhibiting a v h 6 with blocking antibodies.NS398 combined with a v h 6 blocking antibodies did not further reduce VB6 invasion.Because VB6 invasion of Matrigel is a v h 6 dependent (Fig. 2A; ref. 20), COX-2 seems to promote a v h 6 -dependent invasive activity.Inhibition of COX-1 did not affect VB6 invasion (P < 0.19).C1 control cells invaded poorly and were unaffected by these treatments (Fig. 2A).H357 cells lack a v integrins and invade Matrigel using a 3 h 1 -and a 6 -dependent mechanisms (data not shown).Inhibition of COX-2 (NS398 or indomethacin) had no significant effect on H357 invasion (P < 0.424 and P < 0.331, respectively; Fig. 2A).Inhibition of VB6 but not H357 cells by NS398 indicates the effects are a v h 6 specific.
To confirm that suppression of VB6 invasion was not due to growth inhibition.Cells were grown on Matrigel gels (diluted 1:2 in a-MEM) for 72 hours F NS398, extracted using cell dispersal NOTE: Twenty OSCCs, 39 epithelial dysplasias with a minimum of 5 years of clinical follow-up (21 nontransforming and 18 transforming to OSCC), and 14 benign polyps showing fibroepithelial hyperplasia were chosen at random and stained for a v h 6 and COX-2 before being scored according to the Quickscore method (23).Briefly, the staining intensity was scored out of three (1, weak; 2, moderate; 3, strong), and the proportion of the tumor cells staining positively was scored out of four (1, <25%; 2, 25-50%; 3, 51-75%; 4, 76-100%).The score for intensity was added to the score for proportion to give a score in the range of 0 to 7 and grouped as À (score = 0), + (score = 1-3), ++ (score = 4-5), or +++ (score = 6-7).There was significantly less expression of both a v h 6 and COX-2 in hyperplastic or dysplastic epithelium compared with OSCC.Neither protein preferentially identified the subgroup of dysplasias that subsequently transformed.
solution (BD Biosciences), and counted (Casy 1 counter; Sharfe System).No inhibition of growth occurred in H357, VB6, or C1 cells (data not shown: P < 0.448, P < 0.306, and P < 0.581, respectively).Similarly, NS398 had no effect on proliferation of cells on uncoated plastic (data not shown).Because VB6 cells have been genetically manipulated to express high levels of a v h 6 , invasion assays were carried out using three OSCC cell lines (BICR6, CA1, and H157), which express high levels of endogenous a v h 6 and show a v h 6 -dependent invasion in Transwell assays (Fig. 2B).Invasion of all three cell lines was inhibited by NS398 (Fig. 2B; BICR6, P < 0.0001; CA1, P < 0.0001; H157, P < 0.011).
NSAIDS have been reported to have antitumor effects through both COX-2-dependent and COX-2-independent mechanisms (1, 2).To test whether suppression of a v h 6 -dependent invasion by NS398 was modulated specifically through inhibition of COX-2, the invasion assays were repeated following RNAi knockdown of COX-2 protein.Forty-eight hours after RNAi treatment, levels of COX-2 protein were reduced in H357, VB6, and C1 cells by 50%, 50%, and 69%, respectively; the (presumed) 65-kDa unglycosylated form had disappeared completely (Supplementary Fig. S1B).Suppressing COX-2 protein reduced invasiveness of VB6 cells to control C1 levels but had no effect on H357 or C1 invasion (Supplementary Fig. S1B; P < 0.003, P < 0.86, and P < 0.89, respectively).PGE 2 restores A v B 6 -dependent invasion.To determine whether COX-2 modulated a v h 6 -dependent invasion through a metabolic product of COX, invasion assays were repeated, but adding NS398 with PGE 2 , PGF 2a , or U46619 (a thromboxane A 2 mimetic).PGE 2 at 2 to 4 ng/mL completely abrogated the antiinvasive activity of NS398 (Fig. 2C; P < 0.0017), whereas PGF 2a and U46610 had no effect (Fig. 2C; P < 0.444 and P < 0.276, respectively).PGE 2 also seemed to produce some restoration of invasion in the presence of a v h 6 blocking antibodies, although this did not reach significance (P = 0.09; data not shown).
Interestingly, the dose response of VB6 cells to PGE 2 produced a bell-shaped curve, with restoration of invasion between a narrow spectrum of concentrations (2-4 ng/mL) but not at higher concentrations (Fig. 2C).Indeed, in the absence of NS398, PGE 2 at concentrations of z10 ng/mL inhibited invasion of all three cell lines (data not shown).Similar biphasic response curves have been described previously, and it is suggested that this may result from prostaglandins interacting nonspecifically with other classes of prostanoid receptors (27).PGE 2 levels were measured by ELISA (Cayman Chemicals); the cell lines produced similar levels of PGE 2 .NS398 treatment or RNAi knockdown of COX-2 inhibited prostaglandin production by >95%, suggesting that synthesis of the prostaglandin was mediated largely through COX-2 rather than COX-1 (Fig. 2C).
COX-2 inhibition suppresses A v B 6 -dependent invasion in organotypic culture.NS398 consistently inhibited the a v h 6dependent invasion of VB6 cells in Transwell assays.Such assays, however, oversimplify the invasive process because stromal cells interact dynamically with tumor cells in vivo to promote invasion (28).We, therefore, used organotypic cultures containing fibroblasts to measure invasion (26).
We transfected VB6 cells with h 6 RNAi and measured invasion.Because RNAi effects are transient, organotypic cultures were harvested after 6 days (7 days after transfection).Sections were analyzed to generate an invasion index, as described (26).Seven days after transfection, RNAi knockdown of h 6 was f50% (3 days, 91%; 5 days, 54%; data not shown), and VB6 invasion was inhibited by >95% (Fig. 3A and D).As in Transwell assays, the high a v h 6expressing VB6 cells were significantly more invasive than C1 control cells (P < 0.0002) but invaded at similar levels to the invasive, a v -negative H357 cells (Fig. 3B-D; P < 0.07).NS398 Although expression of both proteins was present in some dysplasias (see Table 1), expression did not correlate with malignant transformation and was significantly lower than in established OSCC.Expression in OSCC often was most prominent at the periphery of invading tumor islands (C ).
treatment reduced VB6 invasion significantly by f90% over three experiments (Fig. 3B and D; P < 0.0001).Similar to Transwell assays, addition of 2 ng/mL PGE 2 completely abolished NS398induced inhibition of invasion (Fig. 3B and D).C1 cells, expressing low levels of a v h 6 , invade organotypic cultures poorly (Fig. 3B  and D), but this limited invasion was still inhibited by NS398 (Fig. 3D).NS398 had no effect on the invasion of a v -negative H357 cells (Fig. 3C and D; P < 0.572).
COX-2 inhibition interferes with A v B 6 post-receptor events.COX-2 inhibition might modulate a v h 6 function by reducing cell surface expression, decreasing ligand binding affinity, and/or interfering with a v h 6 signaling.Flow cytometry revealed that Figure 2. A, Transwell invasion of VB6 cells was inhibited by blockade of a v h 6 integrin (10D5, 1:100) and by 20 Amol/L NS398 (P < 0.001).Indomethacin (500 Ag/mL) produced a similar level of inhibition, whereas SC-560 (10 Amol/L) had no effect.H357 and C1 cell invasion were not affected by anti-a v h 6 antibody or any of the NSAIDs.Although VB6 invasion was maximally inhibited by 5 Amol/L NS398, no inhibition of H357 or C1 invasion was seen up to 50 Amol/L NS398.B, fluorescence-activated cell sorting analysis confirmed high and low a v h 6 expression by VB6 and C1 cells, respectively, and high endogenous a v h 6 expression in OSCC cell lines BICR6, CA1, and H157.H357 cells were a v h 6 negative.Transwell invasion of BICR6, CA1, and H157 was inhibited using the anti-a v h 6 antibody 6.3G9 (Biogen Idec).Similar levels of inhibition were achieved using NS398.C, inhibition of VB6 Transwell invasion by NS398 was abrogated completely by PGE 2 at 2 ng/mL (P V 0.0017), whereas PGF 2a and U46619 had no effect.Titration of PGE 2 produced a bell-shaped curve.All cell lines produced similar levels of PGE 2 .Treatment with COX-2 RNAi or 20 and 50 Amol/L NS398 inhibited PGE 2 production by >95%.
there was no effect on surface levels of a v h 6 in VB6 cells following a 72-hour incubation with NS398 (Supplementary Fig. S2A).We next examined the effect of COX-2 inhibition on a v h 6 ligand binding.We have shown previously that adhesion to the LAP in the cell lines is modulated solely through a v h 6 : VB6 cells adhere well; C1 cells adhere poorly; and a v h 6 -negative H357 cells do not adhere to this substrate (29).Inhibition of a v h 6 with 10D5 antibody inhibited adhesion completely (Supplementary Fig. S2B), whereas SC-560, NS398, indomethacin, or inhibitory antibodies against h 1 integrin had no effect (P < 1.00, P < 0.60, P < 0.65, and P < 1.00, respectively).These data suggest that COX-2 does not modulate a v h 6 expression or activity.
A v B 6 ligand binding activates Rac-1.The role of small GTPase Rac-1 in regulating cell migration and cell spreading following integrin-binding are well described (30)(31)(32).In particular, several lines of evidence indicate that Rac-1 may play a critical role in a number of aspects of tumor development (31)(32)(33)(34), and Rac-1 has also been shown to modulate tumor cell invasion (32)(33)(34).Therefore, we examined the effect of a v h 6 ligand binding on Rac-1 activation in VB6 cells using a pulldown assay.Compared with cells in suspension, binding of VB6 cells to LAP resulted in Rac-1 activation (Fig. 4A, i, lanes 3 and 4).Rac-1 activation increased over time and was proportionally greater after 24 hours than 4 hours (Fig. 4A, i, lane 6).This effect was a v h 6 dependent: C1 control cells on LAP showed reduced Rac-1 activation compared with VB6 cells (Fig. 4A, ii, quantified in Fig. 4A, v; 61% reduction; P V 0.0001).Rac-1 activation in VB6 cells was inhibited by 69% following h 6 knockdown with RNAi (Fig. 4A, iii, quantified in Fig. 4A, v; P V 0.0001).
Figure 3. OSCC cells were grown in organotypic culture, then invasion in cytokeratin-stained sections was quantified (25).Three sections spaced at 150 Am were analyzed from each gel.A, cytokeratin staining of a 6-day VB6 organotypic culture following transient transfection with random or h 6 RNAi.Inhibition of a v h 6 suppressed invasion of VB6 cells (>95%, P V 0.001; quantified in D ).Western blotting confirmed knockdown of h 6 protein at 72 hours (f90%).B, cytokeratin staining of a 14-day VB6 organotypic culture following treatment with vehicle, NS398, or NS398 combined with PGE 2 .NS398 inhibited invasion of VB6 cells (>90%, P V 0.001).Treatment of VB6 cells with PGE 2 at 2 ng/mL restored invasion in the presence of NS398.C1 cells were less invasive than VB6 cells.C, cytokeratin staining of a 14-day H357 organotypic culture following treatment with vehicle or NS398.Invasion of H357 cells was observed both in control-and NS398-treated cultures, with no inhibition of invasion by NS398 seen.D, first histogram quantifies invasion of (A).

Second histogram quantifies invasion of (B) and (C).
Constitutively active Rac-1 restores A v B 6 -dependent invasion in the presence of NS398.To confirm that the inhibition of Rac-1 activation by NS398 was responsible for the inhibition of VB6 invasion, we expressed constitutively active Rac-1 in VB6 cells using transient transfection experiments.Over four experiments, cells transfected with V12-Rac-1 in the presence of NS398 had their invasive activity restored to control levels (Fig. 4B; no inhibition of invasion; P < 0.88) but not when transfected with empty vector controls (Fig. 4B; invasion inhibited; P < 0.0001).Additionally, in the presence of NS398, those VB6 cells that had invaded were found to be enriched for V12 Rac-1-GFP (by 42%; data not shown).
COX-2 inhibition suppresses A v B 6 -dependent invasion in vivo.Organotypic cultures can be transplanted onto the back muscle fascia of nude mice where they grow and invade into the host tissue (26).Using this method, VB6 cells produced significantly more invasion than C1 control cells (Fig. 5A and C).Transplants were established for 7 days before commencing treatment, when animals received NS398 (5 mg/kg i.p.) or DMSO every 72 hours for 5 weeks.Tumor take was 100%.
NS398 inhibited the invasion of VB6 cells in vivo by f70% (P < 0.009; Fig. 5A and C), suppression similar to that seen in Transwell assays and in vitro organotypic culture (Figs. 2 and 3B).There was no significant effect of NS398 treatment on the a vnegative H357 cells (P < 0.933; Fig. 5B and C).Because C1 cells invade poorly in vivo, the effect of NS398 on this line was not tested.
Elevated levels of both a v h 6 and COX-2 have (separately) been reported in oral dysplasia (12,13,40), where it is suggested that expression may identify lesions at risk of malignant transformation.We examined 39 dysplasias from patients with a minimum of 5 years of clinical follow-up.We found that neither a v h 6 nor COX-2 expression identified high-risk epithelial dysplasias.Thus, 20% to 30% of dysplasias were positive for one or both proteins.There were positive nontransforming lesions and, conversely, negative lesions that subsequently transformed.Additionally, focal basal expression of a v h 6 and COX-2 was present in several hyperplastic lesions.Although, generally, there was increased expression of a v h 6 and COX-2 in dysplasias compared with hyperplastic epithelium, this was much lower than expression in OSCC; most OSCC showed strong expression of both proteins with expression particularly prominent at the periphery of invading tumor islands.These data suggest that the invasion-promoting effects of a v h 6 and COX-2 primarily may occur in established malignancy.It is possible, however, that negative dysplasias subsequently may express the proteins at a later disease stage, before transformation.
VB6 cells, expressing high levels of a v h 6 , are significantly more invasive than C1 control cells (this work and refs.20,21).Interestingly, a v -negative H357 cells invade as well as VB6 by using a 3 h 1 integrin-and a 6 integrin-dependent mechanisms. 5hus, not all OSCC tumor cell invasion is due to a v h 6 .NS398 inhibition of invasion was specific to VB6 cells, and therefore a v h 6 , with no effect on H357 or C1 cells.Additionally, a v h 6 -dependent invasion of other OSCC lines through Matrigel was inhibited significantly by NS398 (Fig. 2B).
As well as having direct effects on invasion (4, 41-43), NSAIDs may inhibit tumor proliferation and promote apoptosis (2).However, NS398 did not affect growth of our cells at the concentrations used in this study, suggesting that effects of NS398 seem to be via a direct inhibition of a v h 6 activity.
Although NSAIDs have both COX-2-dependent and COX-2independent antitumor effects (1), we show that inhibition of VB6 invasion was COX-2 activity dependent; a v h 6 -dependent invasion was suppressed using COX-2 RNAi and restored, in the presence of NS398, by exogenous PGE 2 .Recently, we developed a quantitative invasion assay using organotypic cultures (26).High a v h 6 -expressing VB6 cells are significantly more invasive than C1 control cells in such cultures, and invasion is a v h 6 dependent.NS398 markedly inhibited VB6 cell invasion by >90%.As with Transwell assays, addition of exogenous PGE 2 (at 2 ng/mL) restored VB6 invasion in the presence of the COX-2 inhibitor.However, NS398 had no effect on H357 invasion.
Although COX-2 and a v h 6 expression correlate in OSCC in vivo, we found no direct evidence that either regulates expression of the other.Antibody blockade of a v h 6 did not affect levels of COX-2 protein in VB6 cells, nor did COX-2 inhibition affect a v h 6 levels or ligand-binding affinity.Rather, NS398 affected a v h 6 post-receptor events.Based on previous observations (18), we examined the role of Rac-1 in the COX-2/a v h 6 -dependent invasion.The small GTPase Rac-1 regulates cell migration and spreading following integrin binding (30) and may also modulate tumor cell invasion (31,32).Unlike Ras, however, mutation of Rac-1 has not been found in human cancer: activation probably occurring through aberrant, or overactive, signaling pathways (33,34).Rac-1 was activated by a v h 6 ligand binding in VB6 cells, and this activation was not transient but was maintained, indeed increased, over a 24-hour time period.It is possible that pathologic up-regulation of a v h 6 results in overactive Rac-1 signaling, leading to prolonged Rac-1 activation.a v h 6 -dependent Rac-1 activation was COX-2 dependent because it was inhibited by NS398 and restored on addition of PGE 2 .Functional studies confirmed that a v h 6 -dependent invasion was mediated through Rac-1.Thus, transfection with constitutively active Rac-1 restored VB6 invasion in the presence of NS398.Additionally, RNAi-mediated Rac-1 knockdown inhibited VB6 invasion to levels comparable with NS398 treatment or a v h 6 blockade, both in Transwells and in organotypic cultures.
In summary, our data suggest that neither a v h 6 nor COX-2 expression predicts malignant transformation of oral dysplasia.However, there is strong coexpression of both proteins in established OSCC, in accord with our proposal that these molecules might interact in promoting invasion.We show, for the first time, that a v h 6 -dependent invasion in vitro and in vivo by OSCC cells requires COX-2-dependent activation of Rac-1, via upregulation of PGE 2 ; abrogating COX-2, thus, blocks a v h 6 -dependent invasion.
Worldwide, OSCC represents about 5.5% of all malignancies (44).Most patients are treated by surgery, and >50% of patients die within 5 years (44).New types of therapy obviously are required, and both COX-2 and a v h 6 have been suggested as possible therapeutic targets.Our data suggest that NSAIDs may inhibit the pro-invasive effect of both proteins.The significant morbidity and mortality rates of OSCC are such that the therapeutic benefit of these drugs should be considered, particularly in a v h 6 -positive tumors.

Figure 1 .
Figure 1.Immunohistochemistry showing representative a v h 6 and COX-2 expression in hyperplastic oral epithelium (A), dysplastic oral epithelium (B ), and OSCC (C ).Although expression of both proteins was present in some dysplasias (see Table1), expression did not correlate with malignant transformation and was significantly lower than in established OSCC.Expression in OSCC often was most prominent at the periphery of invading tumor islands (C ).

Figure 4 .
Figure 4. A, i, binding of VB6 cells to LAP over 4 hours induced Rac-1 activation compared with cells in suspension (S; lanes 3 and 4).Rac-1 activation increased over time and was proportionally greater at 24 hours (lanes 5 and 6).ii, C1 control cells compared with VB6 cells showed significantly less Rac-1 activation on LAP (61% reduction; P = 0 < 0.0001).iii, RNAi h 6 knockdown in VB6 cells significantly reduced Rac-1 activation on LAP (69% reduction; P = <0.0001).iv, NS398 suppressed a v h 6 -mediated Rac-1 activation in VB6 cells on LAP (72% reduction; P = 0.0057).Addition of PGE 2 restored Rac-1 activation in the presence of NS398.v, densitometric analysis of three combined individual experiments confirmed that Rac-1 activation in VB6 cells on LAP was a v h 6 -dependent, and that this was inhibited by NS398 and restored by PGE 2 .B, VB6 Transwell invasion in the presence of NS398 was restored by V12-Rac-1-GFP to f85% of that of empty vector controls.C, Rac-1 RNAi significantly inhibited VB6 Transwell invasion (P < 0.001).Western blot verified protein knockdown.D, RNAi knockdown of Rac-1 inhibited invasion of VB6 cells in 6-day organotypic culture (P < 0.001).Degree of invasion is quantified in the histogram.

Figure 5 .
Figure 5. Organotypic cultures were transplanted into athymic nude (nu/nu ) mice for 6 weeks as described.Test animals were given 5 mg/kg NS398 i.p. every 72 hours, whereas control animals received vehicle (1:100 DMSO in PBS) only.Five sections were cytokeratin stained and analyzed for each in vivo tumor.A, NS398 inhibited invasion of VB6 cells >70% compared with control animals (P < 0.001).Control C1 cells were less invasive than the high-a v h 6 expressing VB6 cells.B, invasion of H357 was seen in both control-and NS398-treated animals, with no inhibition of invasion by NS398 observed (P < 0.933).C, histograms quantifying invasion of the above results.C1 tumors showed little invasion; hence, the effect of NS398 was not tested (NT).Columns, mean; bars, SE.

Table 1 .
a v h 6 and COX-2 expression in epithelial hyperplasia, dysplasia, and OSCC