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Clinical Cancer Research Vol. 9, 3425-3430, August 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Microsomal Prostaglandin E Synthase-1 Is Overexpressed in Head and Neck Squamous Cell Carcinoma1

Erik G. Cohen, Taghreed Almahmeed, Baoheng Du, Dragan Golijanin, Jay O. Boyle, Robert A. Soslow, Kotha Subbaramaiah and Andrew J. Dannenberg2

Departments of Surgery [E. G. C., T. A., J. O. B.], Pathology [R. A. S.], and Urology [D. G.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021, and Department of Medicine, Weill Medical College of Cornell University and Strang Cancer Prevention Center, New York, New York 10021 [B. D., K. S., A. J. D.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated levels of prostaglandin E2 (PGE2) occur in head and neck squamous cell carcinoma (HNSCC) and have been associated with a poor prognosis. Recently, an inducible microsomal prostaglandin E synthase-1 (mPGES) was identified. This enzyme converts the cyclooxygenase product prostaglandin H2 (PGH2) to PGE2. Given the apparent significance of PGE2 in carcinogenesis, it is important to elucidate the mechanisms that account for increased amounts of PGE2 in HNSCC. By immunoblot analysis, mPGES was overexpressed in 11 of 14 (79%) cases of HNSCC compared with adjacent normal tissue. Immunohistochemistry localized mPGES expression to neoplastic epithelial cells. Cell culture was used to determine whether cellular transformation was associated with increased amounts of mPGES. Levels of mPGES protein and mRNA were markedly elevated in HNSCC cell lines (1483 and Ca9-22) versus a nontumorigenic oral epithelial cell line (MSK-Leuk1). Interestingly, treatment of MSK-Leuk1 cells with PGE2 caused both dose- and time-dependent stimulation of cell growth. Each of the four known receptors for PGE2 (E-prostanoid receptor subtypes 1–4) was detected in head and neck squamous mucosa. Taken together, these results suggest that overexpression of mPGES contributes to the increased levels of PGE2 found in HNSCC. Additional studies will be needed to determine whether this enzyme is a bona fide target for anticancer therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased levels of PGE23 have been detected in a variety of malignancies including HNSCC (1 , 2) . Several lines of evidence, beyond the finding of elevated levels of PGE2 in tumors, suggest that PGE2 plays a role in the development and progression of cancer. For example, PGE2 can stimulate cell proliferation and motility while inhibiting apoptosis and immune surveillance (3, 4, 5, 6, 7, 8, 9, 10) . Importantly, PGE2 can also induce angiogenesis at least, in part, by enhancing the production of proangiogenic factors including vascular endothelial growth factor (11 , 12) . Consistent with these findings, higher levels of PGE2 in HNSCC specimens correlated significantly with the occurrence of metastatic disease and increased tumor vascularization (2 , 13) . Recent work in experimental animals has also suggested that PGE2 can promote carcinogenesis. In one study, genetic disruption of the EP2 was found to decrease the number and size of experimental tumors (14) . In other studies, treatment with anti-PGE2 monoclonal antibody inhibited the growth of transplantable tumors including HNSCC (10 , 15) . Given this background, it is important to define the enzymatic pathways that are dysregulated in HNSCC leading to increased amounts of PGE2.

The synthesis of PGE2 from arachidonic acid requires two enzymes that act in sequence. COX catalyzes the conversion of arachidonic acid to PGH2. COX-2, the inducible form of COX, is commonly overexpressed in a variety of solid tumors including HNSCC (16 , 17) . Recently, an inducible human mPGES was identified and characterized (18) . This enzyme converts COX-derived PGH2 to PGE2. Increased levels of mPGES have been detected in several human malignancies (19, 20, 21) , raising the possibility that aberrant mPGES expression could contribute to increased amounts of PGE2 in HNSCC.

In this study, we found that mPGES was commonly overexpressed in HNSCC. Cell culture was used to determine the effects of cell transformation on mPGES expression. As reported previously for COX-2 (22) , cell transformation appears to contribute to the increased amounts of mPGES observed in HNSCC. We also report for the first time that all four PGE2 receptor subtypes are expressed in head and neck squamous mucosa. Taken together, it seems likely that mPGES-derived PGE2 signals through PGE2 receptors and thereby contributes to the progression of HNSCC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Rabbit polyclonal antihuman mPGES antiserum was obtained from Cayman Chemical (Ann Arbor, MI). Mouse anti-ß-actin antiserum was obtained from Oxford Biomedical Research (Oxford, MI). DMEM:Ham’s F-12, fetal bovine serum, gentamicin, amphotericin B, Taq polymerase, and Trizol reagent were obtained from Invitrogen (Carlsbad, CA). Lowry protein assay kits, biotinylated antirabbit IgG antibody, and 3,3'-diaminobenzidine were purchased from Sigma Chemical Co. (St. Louis, MO). Streptavidin-horseradish peroxidase was purchased from DAKO Corp. (Carpinteria, CA). Human EP1, EP3, and EP4 expression vectors were gifts from Dr. Mark Abramovitz (Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Quebec, Canada). Human EP2 expression vector was a gift from Dr. John W. Regan (University of Arizona, Tucson, AZ). Dr. Per-Johan Jakobsson (Karolinska Institute, Stockholm, Sweden) generously provided an expression vector for human mPGES. PCR primers were synthesized by Sigma Genosys (The Woodlands, TX). SYBR Green PCR Master Mix, murine leukemia virus reverse transcriptase, RNase inhibitor, oligo(dT)16, and deoxynucleotide triphosphate were purchased from Applied Biosystems (Foster City, CA). Enhanced chemiluminescence solution was from Perkin-Elmer Life Sciences (Boston, MA). RNeasy Mini-kits were obtained from Qiagen (Valencia, CA).

Patient Samples.
Specimens from patients with previously untreated HNSCC were obtained at the time of resection, in accordance with an institutional review board-approved protocol. Tissue specimens from tumor and adjacent normal- appearing mucosa were frozen in liquid nitrogen and stored at -80°C. HNSCC specimens used for immunohistochemistry were obtained from archived, paraffin-embedded tissue blocks from patients with histologically proven HNSCC.

Cell Culture.
The 1483 cell line was derived from a T2N1M0, American Joint Committee on Cancer stage III well-differentiated squamous cell carcinoma of the retromolar trigone (23) . The Ca9-22 cell line was derived from a patient with squamous cell carcinoma of the gingiva (24) . Both 1483 and Ca9-22 cell lines are tumorigenic in immunocompromised mice (23 , 24) . MSK-Leuk1 was established from a dysplastic leukoplakia lesion adjacent to a squamous cell carcinoma of the tongue. It is nontumorigenic in nude mice and exhibits little anchorage-independent growth (25) . 1483 and Ca9-22 cell lines were maintained in DMEM:Ham’s F-12 with 10% fetal bovine serum, 50 µg/ml gentamicin, and 0.25 µg/ml amphotericin B. MSK-Leuk1 cells were maintained in keratinocyte growth medium supplemented with bovine pituitary extract.

Western Blotting.
Frozen human tissue was thawed in ice-cold lysis buffer [150 mM NaCl, 100 mM Tris (pH 8.0), 1% Tween 20, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml trypsin, and 10 µg/ml leupeptin]. Tissues were sonicated for 3 min on ice and then centrifuged at 10,000 x g for 10 min at 4°C to remove particulate matter. The protein concentration of the supernatant was determined using the method of Lowry et al. (26) . Cell lysates were prepared as described previously (22) . Immunoblot analysis for mPGES and ß-actin was performed using methods described in previous studies (19 , 20) .

Immunohistochemistry.
mPGES immunohistochemistry was performed as described previously (19 , 20) . Neutral buffered formalin-fixed tissue was embedded in paraffin. Tissue sections (4 µm) were prepared using a microtome and mounted on Superfrost/Plus slides. Immunohistochemical analysis was carried out within 24 h of sectioning. Sections were deparaffinized in xylene, rehydrated in graded alcohols, and washed in distilled water. Antigen retrieval was performed by steaming the sections in 10 mM citric acid (pH 6.0) for 30 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Slides were washed in PBS and then blocked for 20 min with 2% BSA. Slides were then incubated with antiserum to mPGES at a 1:750 dilution (2% BSA in PBS) for 18 h at 4°C. Control sections were incubated with mPGES antiserum preabsorbed with a 100-fold excess of blocking peptide or preimmune serum. After being washed three times with PBS, the sections were incubated with biotinylated antirabbit antiserum (1:500 dilution) for 1 h at room temperature. The slides were again washed three times in PBS, and then they were labeled using streptavidin-horseradish peroxidase (1:500 dilution) for 1 h at room temperature. The reaction was visualized using 3,3'-diaminobenzidine. Subsequently, the slides were rinsed in tap water and counterstained with hematoxylin. Finally, the slides were dehydrated with ethanol, rinsed with xylene, and coverslipped.

Real-Time PCR Analysis of mPGES.
Total cellular RNA was isolated from cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Reverse transcription was performed using 1 µg of RNA per 50 µl of reaction. Reaction mixture contained 1x PCR Buffer II, MgCl2 (2.5 mM), deoxynucleotide triphosphate (250 µM each), RNase inhibitor (1 unit/µl), murine leukemia virus reverse transcriptase (2.5 units/µl), and oligo(dT)16 (2.5 µM). Samples were incubated for 10 min at room temperature and then cycled at 42°C for 15 min and 95°C for 10 min.

Real-time PCR was performed using SYBR Green PCR Master Mix with 2 µl of cDNA and 200 nM upstream and downstream primer per 20 µl of reaction (iCycler thermal cycler; Bio-Rad Laboratories, Hercules, CA). Each sample was amplified in duplicate in every experiment. Full-length mPGES was excised from the expression vector, gel-purified, and quantified by absorbance at 260 nm. Serial dilutions over a range of 10,000-fold were used to create a standard curve to establish efficiencies of mPGES and ß-actin amplification and for mPGES quantification. Primer pairs were as follows: (a) mPGES, 5'-TGGAGACCATCTACCCCTT-3' (forward) and 5'-CCACGAGGA AGACCAGGAA-3' (reverse); and (b) ß- actin, 5'-GGCATCCTCACCCTGAAGTA-3' (forward) and 5'-GCCAGATTTTCTCCATGTCG-3' (reverse), encoding products of 99 and 73 bp, respectively. Thermal cycling conditions were 95°C for 10 min; followed by 30 s at 94°C, 30 s at 62°C, and 30 s at 72°C for 40 cycles. Mean efficiency of mPGES and ß-actin amplification was 2.04 and 2.08, respectively. Melting curve analysis and agarose gel electrophoresis confirmed a single PCR product. PCR product was extracted from the gel, and correct sequence identity was confirmed by direct sequencing. Relative mPGES expression was calculated using the following equation: ratio = (EmPGES){Delta}TC (control - sample)/ (Eß-actin){Delta}TC (control - sample) (27) .

Analysis of EP Receptor Expression.
Expression of EP receptors in oral cavity mucosa was analyzed by reverse transcription-PCR. RNA was prepared from frozen tissue using the RNeasy Mini-kit (Qiagen), and cDNA was generated as described above. Primer pairs used were as follows: (a) EP1, 5'-TGGGCCAGCTTGTCGGTAT-3' (forward) and 5'-AGCGCCACCAACACCAG-3' (reverse); (b) EP2, 5'-TCCAATGACTCCCAGTCTGAGG-3' (forward) and 5'-TGCATAGATGACAGGCAGCACG-3' (reverse); (c) EP3, 5'-CAGCTTATGGGGATCATGTG-3' (forward) and 5'-TCCGTGTGTGTCTTGCAGT-3' (reverse); (d) EP4, 5'-CAGATTTGCAGGCCATCC-3' (forward) and 5'-GAGCACTGTCTTTCTCAGGA-3' (reverse); and (e) ß-actin, 5'-GGTCACCCACACTGTGCCCAT-3' (forward) and 5'-GGATGCCAC AGGACTCCATGC-3' (reverse). EP receptor cDNAs were used as positive controls. The identity of each PCR product was confirmed by DNA sequencing.

MTT Assay.
MSK-Leuk1 cells were cultured as described above. Approximately 5 x 102 cells in 100 µl of medium were placed in each well of a 96-well plate. After 24 h of incubation at 37°C, cells were treated with varying concentrations of PGE2. Treatment medium was changed every 48 h. At the end of the treatment period, cells were incubated for 3 h with MTT (0.5 mg/ml) in 100 µl of medium. DMSO (100 µl) was added to each well, and the cells were incubated at 37°C for 30 min. Absorption at 560 nm was read using a 96-well plate spectrophotometer (SLT Lab Instruments A-5082).

Statistics.
Comparisons between groups were made using Student’s t test. A difference between groups of P < 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunoblot analysis was used to assess amounts of mPGES in 14 paired samples of HNSCC. Overall, increased amounts of mPGES were detected in 11 of 14 (79%) cases of HNSCC (Fig. 1)Citation . Expression of mPGES protein was undetectable in most samples of nontumorous tissue. Immunohistochemistry was carried out to determine the cellular source of mPGES. Granular cytoplasmic staining for mPGES was detected in neoplastic epithelial cells in HNSCC (Fig. 2)Citation . Immunoreactivity was distributed throughout the tumor. This staining was specific for mPGES because immunoreactivity was lost when the mPGES antiserum was preincubated with a mPGES-blocking peptide.



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  		Fig. 1. mPGES protein is overexpressed in HNSCC. Immunoblot of paired tumorous (T) and nontumorous (NT) tissue from four subjects is shown. Equal amounts of tissue lysate protein (100 µg/lane) were loaded onto a 12.5% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Purified mPGES protein was used as a standard (Std.). The immunoblot was probed sequentially for mPGES and ß-actin.

 


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  		Fig. 2. mPGES is expressed in malignant epithelial cells in HNSCC. Strong diffuse cytoplasmic immunoreactivity for mPGES was found in malignant epithelial cells (x400).

 
Based on the discovery that mPGES was commonly overexpressed in neoplastic epithelial cells in HNSCC, we investigated whether increased amounts of mPGES would be detected in cell lines derived from HNSCC. To address this question, levels of mPGES protein and mRNA were compared in tumorigenic cell lines derived from HNSCC (1483 and Ca9-22) versus a nontumorigenic cell line derived from a premalignant oral lesion (MSK-Leuk1). As shown in Fig. 3, A and BCitation , amounts of mPGES protein and mRNA were markedly increased in the transformed cell lines, consistent with the increased levels of mPGES observed in HNSCC.



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  		Fig. 3. Cell transformation is associated with increased amounts of mPGES protein and mPGES mRNA. A, cellular lysate protein (100 µg/lane) from MSK-Leuk1, 1483, and Ca9-22 cells was loaded onto a 12.5% SDS-polyacrylamide gel; electrophoresed; and subsequently transferred onto nitrocellulose. The immunoblot was sequentially probed with antibodies specific for mPGES and ß-actin. Purified mPGES protein was used as a standard (Std.). B, 0.04 µg of total cellular RNA was reverse transcribed and amplified by real-time PCR using specific mPGES primers with SYBR Green fluorescence. ß-Actin served as an internal control. Columns, means; bars, SD; n = 4. *, P < 0.05; **, P < 0.01. Higher levels of mPGES protein (A) and mPGES mRNA (B) were detected in HNSCC cell lines (1483 and Ca9-22) than in the immortalized nontumorigenic MSK-Leuk1 cell line.

 
One potential mechanism by which PGE2, the product of mPGES activity, could impact on tumor growth is by stimulating cell proliferation. Hence, we next investigated the effects of exogenous PGE2 on the growth of MSK-Leuk1 cells. As shown in Fig. 4ACitation , treatment with 500 nM PGE2 stimulated cell growth more than treatment with 100 nM PGE2. Higher concentrations of PGE2 did not lead to a further increase in cell growth. The growth-stimulatory effects of PGE2 were also time dependent. Cell number was enhanced by approximately 40% after treatment with 500 nM PGE2 for 10 days (Fig. 4B)Citation .



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  		Fig. 4. PGE2 stimulates the growth of oral epithelial cells. MSK-Leuk1 cells were treated with PGE2 for varying lengths of time. A, the effects of 100 or 500 nM PGE2 on cell growth were determined over a 6-day period. Treatment with 500 nM PGE2 stimulated cell growth more effectively than treatment with 100 nM PGE2. B, treatment with 500 nM PGE2 caused time-dependent increases in cell growth over a 10-day period. At the end of 10 days, treatment with PGE2 caused an approximately 40% increase in cell number as determined by MTT assay. Data (mean ± SD; n = 6) are expressed as relative absorbance with the number in the control group kept constant at 1.0 for all time points. *, P < 0.05; **, P < 0.01 versus untreated (control) cells.

 
PGE2 elicits cellular responses via interaction with four cell surface receptors, EP1–4. Reverse transcription-PCR was carried out to determine which receptors were expressed in normal head and neck mucosa. Each of the four known EP receptors was detected (Fig. 5)Citation .



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  		Fig. 5. EP1–4 are expressed in head and neck squamous mucosa. Total cellular RNA was extracted from three samples of normal oropharyngeal mucosa. RNA was reverse transcribed, and cDNA was subjected to 35 cycles of PCR with specific primers for EP1–4. Samples were electrophoresed on a 2% agarose gel with ethidium bromide and photographed under UV light. Standards (Std.) are full-length cDNA expression vectors for each EP receptor. No bands were observed when cDNA was omitted from the PCR reaction, or when reverse transcriptase enzyme was not included in the reverse transcription reaction (data not shown).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we found that mPGES was overexpressed in approximately 80% of cases with HNSCC. This finding is consistent with other recent reports describing increased expression of mPGES in cancers of the lung, colon, and endometrium (19, 20, 21) . It is likely, therefore, that enhanced expression of mPGES in addition to COX-2 contributes to the increased amounts of PGE2 detected in HNSCC. Importantly, multiple lines of evidence suggest that overproduction of PGE2 is mechanistically linked to the progression of HNSCC. Higher levels of PGE2 in human HNSCC specimens correlated significantly with increased tumor vascularization and the occurrence of metastatic disease (2) . In a recent study that used an experimental model of human HNSCC, treatment with a selective COX-2 inhibitor markedly decreased the rate of tumor growth in association with a significant reduction in intratumoral levels of PGE2 (15) . Importantly, treatment with a monoclonal antibody to PGE2 was as effective as the selective COX-2 inhibitor in reducing tumor growth (15) . Although these findings firmly established the significance of PGE2 as a determinant of tumor growth, the precise mechanism is uncertain. In this study, we demonstrate that PGE2 can directly stimulate cell growth. Others have shown that PGE2 can stimulate angiogenesis (11) while enhancing the survival (3) and motility (4) of cancer cells. Immune surveillance is inhibited by PGE2 (6, 7, 8, 9, 10) . The relative importance of these different mechanisms is uncertain and may well vary in different tumor types or stages of disease.

PGE2 exhibits biological activity through binding to G-protein-coupled receptors. Four PGE2 receptors, EP1–4, have been identified. Because of the significance of PGE2 in carcinogenesis, the potential role of individual receptors is being actively investigated. Different PGE2 receptors appear to be important in carcinogenesis in different tumor models and tumor types (14 , 28, 29, 30) . We show for the first time that all four EP receptors are expressed in human oral squamous mucosa. Future experiments will be needed to define which EP receptor(s) is important for mediating the growth stimulating effects of PGE2.

Experiments were also carried out to begin to elucidate the mechanism(s) that accounts for overexpression of mPGES in HNSCC. Markedly increased amounts of both mPGES mRNA and mPGES protein were detected in HNSCC tumor cell lines versus a nontumorigenic cell line derived from a premalignant oral lesion. This result suggests that cellular transformation is one of the mechanisms leading to enhanced expression of mPGES in HNSCC. mPGES can be induced by cytokines including interleukin 1ß and tumor necrosis factor {alpha} (18, 19, 20 , 31 , 32) . Hence, it seems highly likely that multiple mechanisms will contribute to the overexpression of mPGES in HNSCC. Additional experiments are warranted to define the signaling mechanisms that regulate the expression of mPGES.

The COX enzymes produce PGH2, which, in turn, is metabolized to a variety of eicosanoids. One of the products, PGE2, has procarcinogenic properties, but other products, such as prostacyclin, inhibit carcinogenesis (33) . Because inhibiting COX suppresses the synthesis of both procarcinogenic and anticarcinogenic eicosanoids, more selective forms of treatment may prove useful. Examples of more selective treatment would include a selective inhibitor of PGE2 synthesis or an EP receptor antagonist. Initial studies have suggested that mPGES represents a potential therapeutic target. More specifically, cells overexpressing mPGES and COX-2 produced more PGE2, grew faster, and exhibited abnormal morphology compared with cells in which either COX-2 or mPGES was overexpressed (34) . To further investigate whether mPGES is a therapeutic target, it will be important to determine whether overexpressing or knocking out mPGES affects tumor formation or growth. The availability of selective inhibitors of mPGES would permit complementary experiments to be done. Importantly, our observation that mPGES is commonly overexpressed in HNSCC provides the basis for future studies that will evaluate whether mPGES is a bona fide therapeutic target.


   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.

1 Supported by NIH Grants T32 CA09685 and 1 R01 CA82578. Back

2 To whom requests for reprints should be addressed, at New York Presbyterian Hospital-Cornell, 525 East 68th Street, Room F-206, New York, NY 10021. Phone: (212) 746-4403; Fax: (212) 746-4885; E-mail: ajdannen{at}med.cornell.edu Back

3 The abbreviations used are: PGE2, prostaglandin E2; HNSCC, head and neck squamous cell carcinoma; mPGES, microsomal prostaglandin E synthase-1; PGH2, prostaglandin H2; EP, E-prostanoid receptor; COX, cyclooxygenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back

Received 12/27/02; accepted 2/28/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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