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10.1245/s10434-006-9001-4
Annals of Surgical Oncology 13:1224-1234 (2006)
© 2006 Society of Surgical Oncology
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Original Article

Clinical Implications of Microsomal Prostaglandin E Synthase-1 Overexpression in Human Non–Small-Cell Lung Cancer

Hao-Wei Wang, MD1, Chung-Tsen Hsueh, MD, PhD2, Chien-Fu Jeff Lin, MD, PhD3, Teh-Ying Chou, MD, PhD4, Wen-Hu Hsu, MD1, Liang-Shun Wang, MD1 and Yu-Chung Wu, MD1

1 Division of Thoracic Surgery, Department of Surgery, Taipei Veterans General Hospital and School of Medicine, National Yang-Ming University, 201, Sec 2, Shih-Pai Road, Taipei 112, Taiwan
2 Division of Hematology/Oncology, Department of Internal Medicine, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA
3 Department of Statistics, National Taipei University, 151 University Road, San Shia, Taipei 237, Taiwan
4 Department of Pathology, Taipei Veterans General Hospital and School of Medicine, National Yang-Ming University, 201, Sec 2, Shih-pai Road, Taipei 112, Taiwan

Correspondence: Address correspondence and reprint requests to: Yu-Chung Wu, MD; E-mail: wuyc{at}vghtpe.gov.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Microsomal prostaglandin E synthase-1 (mPGES-1) has recently been found to overexpress in human cancers, including non–small-cell lung cancer (NSCLC). However, the clinical value is largely unknown. The aim of this study was to investigate the associations between mPGES-1 expression in NSCLC and the clinical characteristics and survival outcome.

Methods: Between 2001 and 2003, paired fresh tumorous and nontumorous samples were prospectively procured from patients undergoing surgery for NSCLC. The expression of mPGES-1 was assessed by using Western blot in 93 subjects and reverse transcriptase-polymerase chain reaction in 35. Overexpression of mPGES-1 was defined as a more than 2-fold expression in the tumorous sample compared with the corresponding nontumorous one. Immunohistochemistry was used to confirm its localization to the tumor cells. In a subset of 30 cases, cyclooxygenase-2 (COX-2) was also analyzed to assess its association with mPGES-1.

Results: The protein and messenger RNA of mPGES-1 were both expressed at higher levels in the tumor samples (P < .001 and P = .006, respectively). The expressions of mPGES-1 and COX-2 were unrelated (P = .715). Overexpression of mPGES-1 protein was observed in 61 (65.6%) of 93 patients, but it was not significantly associated with the clinicopathologic characteristics or overall and disease-free survivals. However, mPGES-1 overexpression seemed to be associated with the likelihood of subsequent pulmonary metastases and a lower tendency for developing bony metastases (P = .001 and P = .006, respectively).

Conclusions: Our results demonstrated that mPGES-1 was overexpressed in NSCLC, unassociated with COX-2. Overexpression of mPGES-1 per se was not a prognostic indicator, but it might be implicated in the organ preference of metastasis.

Key Words: Non–small-cell lung cancer • Prostaglandin-endoperoxide synthase • Prostaglandins • Cyclooxygenase inhibitors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lung cancer is the leading cause of cancer-related deaths in many countries, including Taiwan.1 Despite improvements in diagnosis and treatment, the overall 5-year survival rate remains <15%.2 Novel molecular targets regarding prevention and treatment will be crucial to improve this poor outcome. During the past few decades, there has been an enormous amount of new information regarding the roles of the prostaglandin (PG) biosynthetic pathway in human cancers. The interest in these studies was elicited by several epidemiological reports indicating that chronic use of nonsteroidal anti-inflammatory drugs is effective in reducing the risks of several cancers.3 Cyclooxygenase (COX), the key enzyme in the biosynthesis of PGs, is the main target of nonsteroidal anti-inflammatory drugs. Two isoforms of COX, COX-1 and COX-2, each encoded by separate genes, have been identified.

In the biosynthesis of PGs, arachidonic acid is first mobilized from membrane glycerophospholipids by the action of phospholipase A2. The COX enzymes then catalyze the formation of an intermediate PGG2, followed by reduction to PGH2. PGH2 is subsequently converted to several structurally related PGs, including PGE2, PGD2, PGF2{alpha}, PGI2, and thromboxane A2, by the activity of specific PG synthases.

Among several kinds of PGs, increased production of PGE2 has been found in various malignancies.4–6 Further experimental studies indicated that PGE2 might play a key role in carcinogenesis and disease progression.7–10 PGE synthase (PGES), which catalyzes the formation of PGE2, was first identified by Jakobsson et al.11 in 1999. Recent advances in this field have led to the identification of at least three PGES isoenzymes: namely, cytosolic PGES, microsomal PGES (mPGES)-1, and mPGES-2.12–15 Similar to COX-1, cytosolic PGES was reported to be constitutively expressed in many tissues and functionally coupled with COX-1 in the maintenance of tissue homeostasis,12 whereas mPGES-1 was found to be inducible, to act in concert with COX-2, and to contribute to a variety of physiological and pathologic conditions, such as fever, inflammation, and reproduction.14,16,17

Increased expression of COX-2 has been found in a variety of human malignancies, including lung cancer,18–20 and several studies have suggested it as an indicator of poor prognosis for lung cancers.21,22 Numerous trials were designed to test selective COX-2 inhibitors in the prevention and adjuvant therapy of cancers. Some studies showed promising results.23 Unfortunately, recently released data from major postmarketing multicenter trials disclosed a significant increase in the incidence of cardiovascular adverse events among users of COX-2 inhibitors.24,25 Consequently, some of the clinical trials on the use of COX-2 in cancer prevention and treatment were halted. Suppressed formation of PGI2 in endothelial cells was postulated to be linked to the COX-2–associated cardiovascular risk.25 Therefore, mPGES-1, which resembles COX-2 as an inducible enzyme and acts downstream to COX-2 on the synthesis of PGE2, would theoretically be a more specific and rational target for blockade of PGE2 formation in numerous pathologic conditions. Recently, overexpression of mPGES-1 was reported in non–small-cell lung cancer (NSCLC).26,27 However, its clinical significance has not been properly investigated and currently warrants elucidation.

In this prospective study, we procured samples from surgical specimens of NSCLC patients. The expression of mPGES-1 in the tumor and nontumor tissue was determined by immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR). The aim of this study was to investigate the relationships between mPGES-1 expression in NSCLC and the clinical characteristics and survival outcome.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
A total of 93 patients with NSCLC who underwent surgical resections in our institute between March 2001 and June 2003 were enrolled in this study. The patients included 68 (73.1%) men and 25 (26.9%) women, with a mean age of 66.3 ± 11.4 years (mean ± SD; range, 30–83 years). Tumor histological characteristics and stages were classified according to the World Health Organization classification28 and the current international staging system for lung cancer.29 Sixty tumors were classified as adenocarcinoma, 22 as squamous cell carcinoma, and 7 as large-cell carcinoma. A lobectomy was performed in 72 patients (77.4%), bilobectomy in 9 (9.7%), pneumonectomy in 5 (5.4%), segmentectomy in 3 (3.2%), and wedge resection in 4 (3.4%). Radical mediastinal lympha-denectomy was performed in 91 patients (97.8%), and the mean number of removed lymph nodes was 25.5 ± 13.2 (mean ± SD) per person. After surgery, the patients were regularly followed up with systemic history-taking and physical examinations, serum tumor markers, computed tomography or radiography of the chest, abdominal sonography, and bone scintigraphy at 3-month intervals. The study follow-up ended in July 2005, and the median follow-up duration was 32.2 months (range, 17.4–47.8 months).

Tissue Procurement
In the study subjects, lung tumor and the corresponding healthy lung tissue were procured from the resected specimen at the time of operation. The samples were obtained from a nonnecrotic area of the tumor and from adjacent nontumorous tissue. The tissue was immediately placed in cryovials, frozen in liquid nitrogen, and stored at –80°C until analysis. Some of the tissue was directly fixed in neutral buffered formalin and then embedded in paraffin for immunohistochemical study. The tissue procurement protocol was approved by the institutional review board, and written informed consent was obtained from all patients.

Tissue Preparation and Protein Extraction
Tissue protein extraction was performed as previously described,30 with some modifications. In brief, frozen tissue was homogenized and thawed in ice-cold radioimmunoprecipitation buffer with 100 µg/ mL of phenylmethylsulfonyl fluoride, 25 µg/mL of aprotinin, 25 µg/mL of leupeptin, 10 µg/mL of soybean trypsin inhibitor, and 1 mM sodium orthovanadate. The lysate was left on ice for 20 minutes and then centrifuged at 12,000 rpm for 10 minutes. The clarified supernatant was collected, and the protein concentration was measured by using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).

Western Blotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as previously described.31 Fifty micrograms of protein from each sample was run in sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using a Bio-Rad Mini-Protean system with an 8% resolving gel and 4% stacking gel. The resolved proteins were transferred onto Immobilon polyvinyl difluoride membranes (Millipore Corporation, Bedford, MA). Ponceau S (Sigma Chemical, St. Louis, MO) staining of the membranes was performed to assess the equivalence of sample loading and gel transfer. Computer densitometry was used to determine the relative loading. The membranes were then destained with tap water for several washes. After blocking with 5% skim milk in Tris-buffered saline containing .1% Tween 20, the membranes were incubated with rabbit anti-human mPGES-1 polyclonal antibody (1/500; Cayman Chemical, Ann Arbor, MI) and goat anti-human COX-2 polyclonal antibody (1/1000; Santa Cruz Biotechnology, Santa Cruz, CA), respectively. The blots were then incubated with anti-rabbit horseradish peroxidase–conjugated secondary antibody for mPGES-1 (1/2000; Amersham Pharmacia Biotech, Buckinghamshire, UK) or anti-goat antibody for COX-2 (1/5000; Santa Cruz).

Subsequently, membranes were developed by using the Pierce SuperSignal chemiluminescent detection reagents (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions and exposed to NEN Renaissance X-ray film (New England Nuclear, Boston, MA) with intensifying screens. The linear-range signal intensity of each specific band on the fluorogram was quantitated by a densitometric scanning system, and comparison of proteins of interest was performed after normalization to the densitometric scanning of the Ponceau S staining. The control value of Ponceau S was assigned an arbitrary unit of 1, and the expression of each protein was denoted as arbitrary densitometry units (ADUs) relative to the corresponding value of the Ponceau S stain. Overexpression of a specific protein was defined as a 2-fold increase of the ADUs in the tumor sample compared with the nontumor sample.

RNA Isolation and Reverse Transcription
Frozen tissue was homogenized, and total RNA was isolated by using RNeasy Mini Kits (Qiagen, Santa Clarita, CA). A maximum of 600 ng of total RNA was reverse-transcribed by using a GeneAmp RNA PCR Kit (PerkinElmer, Foster City, CA) according to the manufacturer’s protocol.

Polymerase Chain Reaction
Aliquots of complementary DNA (cDNA) were used in PCR for mPGES-1, COX-2, and ß-actin (as an internal control) with the previously published primer sequences and reaction conditions.32–34 The PCR protocol consisted of an initial denaturation of cDNA at 94°C for 5 minutes, followed by 27 cycles (35 cycles for COX-2 and ß-actin) of amplification: denaturing at 94°C for 45 seconds (95°C and 1 minute for COX-2 and ß-actin), hybridizing at 65°C for 45 seconds (51°C and 30 seconds for COX-2; 58°C and 30 seconds for ß-actin), elongating at 72°C for 45 seconds (2 minutes for COX-2 and ß-actin), and extension at 72°C for 10 minutes (7 minutes for COX-2 and ß-actin). The primer sequences were as follows: mPGES-1 (344 base pairs) sense, 5'-CTC TGC AGC ACG CTG CTG G-3', and antisense, 5'-GTA GGT CAC GGA GCG GAT GG-3'; COX-2 (304 base pairs) sense, 5'-AGT CAA AGA TAC TCA GGC AGA-3', and antisense, 5'-GTA GTT CTG GGT CAA ATT TCA-3'; ß-actin (544 base pairs) sense, 5'-CAG CTC ACC ATG GAT GAT GAT A-3', and antisense, 5'-CCA GAC GCA GGA TGG CAT-3'.

The PCR product was resolved by electrophoresis on 2% agarose gel in Tris–acetate–ethylenediamine-tetraacetic acid buffer and visualized with ethidium bromide staining of the gel. The signal intensity of each specific band was quantitated by a densitometric scanning system. Beta actin was used as a loading control. The expression of each messenger RNA (mRNA) was denoted as ADUs relative to the corresponding densitometry value of ß-actin.

Immunohistochemistry
Neutral buffered formalin-fixed tissue was embedded in paraffin. Tissue sections (4 µm) were prepared by using a microtome and mounted on SuperFrost Plus slides (Dako, Kyoto, Japan). Sections were deparaffinized in xylene, rehydrated in graded alcohol, and washed in distilled water. Antigen retrieval was performed by steaming the sections in 10 mM citric acid (pH 6.0) for 30 minutes. Subsequently, endogenous peroxidase activity was blocked with 3.0% hydrogen peroxide. The slides were washed three times in phosphate-buffered saline and blocked for 20 minutes with 3% bovine serum albumin in phosphate-buffered saline. Tissue sections were then incubated with rabbit anti-human mPGES-1 polyclonal antibody (Cayman) at a 1/50 dilution for 1 hour at room temperature. Control sections were incubated with mPGES-1 antiserum preabsorbed with a 100-fold excess of mPGES-1 blocking peptide (Cayman) as previously described by Yoshimatsu et al.26 After being washed three times with phosphate-buffered saline, the secondary antibody Dako Link (Dako LSAB2 kit) was applied for 20 minutes and then rinsed with Tris-buffered saline. Additional washing was followed by incubation with streptavidin horseradish peroxidase (Dako LSAB2 kit) for 20 minutes. Immunoreactivity was visualized by incubation of sections with 3-amino-9-ethylcarbazole (Sigma). Subsequently, the slides were rinsed in tap water and counterstained with hematoxylin. The slides were then dehydrated with ethanol, rinsed with xylene, and mounted.

Statistical Analyses
Between paired tumor and nontumor samples, the ADUs of mPGES-1 protein and mRNA expression were compared by using the Wilcoxon signed rank test. The association between the expression of COX-2 and mPGES-1 in tumor tissue was analyzed with Fisher’s exact test. The associations between mPGES-1 overexpression and various clinicopathologic parameters were analyzed with Student’s t-test and Fisher’s exact test.

The recurrence patterns were categorized as loco-regional, lung, bone, liver, brain, and other metastases according to the first documented relapse site. There were overlaps among patients within different categories because one may have the first relapse at more than one site. Locoregional recurrence was defined as tumor recurrence in the surgical margin, including the bronchial stump, pleura, and chest wall and the hilar and mediastinal lymph nodes, or the development of pleural seeding and malignant pleural effusion. Tumor recurrences in the contralateral lung, bilateral lungs, or unequivocally within different lobes of the ipsilateral lung were regarded as pulmonary metastases.

Disease-free survival was measured from the date of operation to the date of the first documented recurrence or to the date of last follow-up if no recurrence had occurred. Survival analyses were conducted by using the Kaplan-Meier method and univariate Cox proportional hazards model. A P value of <.05 was considered statistically significant. All analyses were performed with SPSS software, version 12.0 (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Microsomal PGES-1 Was Overexpressed in NSCLC Tumor Samples
Western blot was performed in 93 paired samples and RT-PCR was performed in 35 to assess the expression of mPGES-1. Figure 1Go demonstrates the representative immunoblots and RT-PCR and the semiquantitative results. The protein and mRNA of mPGES-1 both exhibited significantly higher expression in the tumor tissue than in the corresponding nontumorous samples (P < .001 and P = .006, respectively). Overexpression of mPGES-1 was observed in 61 patients (65.6%), and immunohistochemistry (IHC) stains confirmed that mPGES-1 was localized in the tumor cells rather than the stromal tissue (Fig. 2Go). The IHC stain was considered specific for mPGES-1 because the immunoreactivity was lost when the antiserum to mPGES-1 was preincubated with mPGES-1 blocking peptide.


Figure 1
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  		FIG. 1. The protein and messenger RNA (mRNA) of microsomal prostaglandin E synthase (mPGES)-1 were overexpressed in non–small-cell lung cancer tumor tissue. (A) Representative immunoblots of paired nontumor (N) and tumor (T) tissues from five subjects. The mPGES-1 level was increased in five samples, and cyclooxygenase (COX)-2 was increased in three. Ponceau S stains were used as a loading control. (B) Boxplot showing the semiquantitative results of the mPGES-1 protein levels (in arbitrary densitometry units; ADU) in paired tumor and nontumor tissues (n = 93). Boxes indicate 25th, 50th, and 75th percentiles. A significantly higher protein level was observed in tumor tissue than in the corresponding nontumor samples (P < .001 by Wilcoxon signed rank test). (C) Representative reverse transcriptase-polymerase chain reaction for mPGES-1 and COX-2 in five paired nontumor (N) and tumor (T) samples. Beta actin was used as an internal control. (D) Boxplot showing that the mPGES-1 mRNA level was significantly higher in tumor tissue (n = 35; P = .006 by Wilcoxon signed rank test).

 

Figure 2
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  		FIG. 2. Representative results of microsomal prostaglandin E synthase (mPGES)-1 expression on tissue sections of (A) primary lung squamous cell carcinoma and (B) adenocarcinoma by immunohistochemical (IHC) staining. If Western blot showed overexpression of mPGES-1, IHC was performed to confirm its localization. Diffuse cytoplasmic immunoreactivity for mPGES-1 was seen in the cancer cells. Brown indicates positive staining of mPGES-1; blue indicates counterstaining with hematoxylin (original magnification, x200).

 
The Expressions of mPGES-1 and COX-2 Were Not Associated
In a subset of 30 pairs, the level of COX-2 was determined. COX-2 overexpression was found in 15 patients (50%). No significant association existed between the expression of COX-2 and mPGES-1 (P = .715; Table 1Go).


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  		TABLE 1. The association between COX-2 and mPGES-1 protein expression in 30 paired samples
 
Overexpression of mPGES-1 Protein Has No Prognostic Relevance
The relationships between the status of mPGES-1 protein expression and various clinicopathologic characteristics are listed in Table 2Go. No significant association existed between mPGES-1 protein expression and clinical characteristics including age, sex, smoking index, histological cell type, cell differentiation, evident angiolymphatic invasion, and tumor staging. The overall and disease-free survival curves with regard to mPGES-1 protein expression are depicted in Fig. 3Go. Overexpression of mPGES-1 protein was not significantly associated with overall or disease-free survival (P = .718 and P = .870, respectively). In contrast, tumor differentiation grade (P = .049), pT stage (P = .025), pN stage (P < .001), and overall stage (P < .001) were positive prognostic factors regarding the overall survival (Table 3Go). The last three factors were also significant prognostic indicators in disease-free survival analysis (P = .001, P < .001, and P < .001, respectively).


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  		TABLE 2. Relationship between clinicopathologic characteristics and mPGES-1 expression
 

Figure 3
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  		FIG. 3. Kaplan-Meier survival curves showing (A) cumulative overall survival and (B) disease-free survival with regard to the expression profile of microsomal prostaglandin E synthase (mPGES)-1. No significant difference in overall survival and disease-free survival was observed between mPGES-1 (+) and mPGES-1 (–) patients (P = .718 and P = .870, respectively, by univariate Cox proportional hazards model).

 

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  		TABLE 3. Univariate Cox’s proportional hazards model for survival analysis
 
Microsomal PGES-1 Expression Might Be Related to the Patterns of Tumor Recurrence
During the follow-up period, 40 patients (43.0%) developed tumor recurrences, including 15 locoregional recurrences and 19 lung, 13 bone, 4 liver, 3 brain, and 4 other metastases. The recurrence patterns with regard to mPGES-1 expression are listed in Table 4Go. Overexpression of mPGES-1 was observed in 17 (89.5%) of the 19 patients who subsequently developed lung metastasis, whereas only 4 (30.8%) of 13 patients who developed bony metastasis exhibited mPGES-1 overexpression in their original tumor samples (P = .001 and P = .006, respectively).


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  		TABLE 4. Relationship between mPGES-1 expression and failure patterns
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Since its first identification in 1999,11 PGES has been found to be involved in various pathophysiological events.16 Subsequent studies disclosed increased expression of mPGES-1 in cancers of the lung, colon, stomach, head and neck, endometrium, and penis.26,27,35–39 Yoshimatsu and colleagues26 first demonstrated overexpression of mPGES-1 in NSCLC. However, that study was conducted on a relatively small sample size, in which an increased level of mPGES-1 was detected in 15 (80%) of 19 cases. Ermert and collaborators27 later also observed increased expression of mPGES-1 in NSCLC by using IHC analysis. The clinical significance of mPGES-1 overexpression in cancer was not well understood. Endoh and colleagues40 identified eight genes, including PGES, that would jointly predict the prognosis of patients with pulmonary adenocarcinoma. However, the detailed clinical data were not provided, and the contributions of individual genes were not assessed in that study.

In this study, we prospectively collected 93 paired specimens and demonstrated overexpression of mPGES-1 in 65.6% of NSCLC tumor samples by Western blot analysis. Increased transcription of mPGES-1 was evidenced by RT-PCR. We used IHC merely to confirm the protein’s localization to tumor cells. Assessing the extent of protein expression by Western blot instead of IHC could avoid the bias originating from the intrinsic subjectivity during the evaluation of IHC results, which has commonly been questioned, and it would theoretically yield a more reliable result.

The observation that mPGES-1 was overexpressed in NSCLC tumor tissue suggests that mPGES-1 may play a role in early tumorigenesis; this has been supported by several lines of evidence from different experimental systems. Murakami et al.14 showed that transfection of mPGES-1 in combination with COX-2 into human embryonic kidney 293 cells resulted in a marked increase in the production of PGE2 in response to stimulation with A23187 (immediate response) or interleukin 1 (delayed response) and, moreover, that the cotransfected human embryonic kidney 293 cells exhibited faster proliferation and aberrant morphology. Additionally, Kamei and colleagues41 demonstrated that transfection of the C-terminal mPGES-1 cDNA into human colon cancer HCA-7 cells could facilitate the growth of HCA-7 cells, whereas treating the cells with an mPGES-1–specific antisense oligonucleotide resulted in a reduction of cell growth. These studies suggest that mPGES-1 contributes to in vitro tumorigenesis through increased production of PGE2. Conversely, evidence suggests that PGI2 may play an antineoplastic role by suppressing inflammation42 or by preventing metastasis.43 Keith et al.44 showed that transgenic mice with selective pulmonary overexpression of prostacyclin synthase were protected from lung cancer when exposed to distinct carcinogenesis protocols.

These studies suggested that shifts in the balance between the production of "good" PG (PGI2) and "bad" PG (PGE2) may play a role in promoting tumor development and progression. However, in this study we demonstrated no significant association between mPGES-1 overexpression and any of the clinicopathologic characteristics, including the histological types, tumor staging, and overall or disease-free survival. This result seems somewhat discordant to the above-mentioned hypothesis because if the hypothesis were true, then overexpression of mPGES-1 would be expected to tip the PG balance toward the procarcinogenic end and contribute to disease progression. One of the possible explanations for this seemingly negative result is that overexpression of mPGES-1 in tumors may not necessarily correspond to an increased level of PGE2 production in vivo. In a recent study by Blaine et al.,45 transgenic mice were created with selective pulmonary overexpression of mPGES-1, and the mice were exposed to a complete carcinogen protocol. It showed that the level of mPGES-1 overexpression in lung tumors from the transgenic mice did not correlate with the level of PGE2 produced. It was postulated that COX-2 induced in tumors is limiting for PGE2 production, and, therefore, increases in mPGES-1 expression do not result in comparable increases in PGE2 production.

Another possible reason why mPGES-1 overexpression did not correlate with clinical outcomes is that there might be a ceiling effect for the role of PGE2 in lung tumorigenesis. There has been evidence that cellular transformation could enhance mPGES-1 expression in NSCLC.26 In the above-mentioned transgenic mice study by Blaine et al.,45 exposure to carcinogens resulted in increased mPGES-1 expression in lung tumors from both transgenic and non-transgenic mice. However, the transgenic mice did not exhibit a significant difference in tumor formation. It was thus hypothesized that there might be an upper threshold for a PGE2 effect above which no additional effects are seen. The increased levels of PGE2 production caused by the carcinogens themselves may have reached this threshold, and further increases in mPGES-1 expression may therefore have had no additive effect.

The regulation of mPGES-1 is multifold and involves several factors, including ret/PTC, early growth response-1, Ras, nuclear factor-{kappa}B, and mitogen-activated protein kinase (MAPK) pathways.26,46–51 The nuclear factor-{kappa}B, Ras, and MAPK pathways also contribute to the regulation of COX-2, as shown by several studies.49,52–54 Coordinate upregulation of mPGES-1 and COX-2 was found in various conditions, including fever,17 inflammation,14,55 and atherosclerosis.56 However, data regarding the association between the expression of mPGES-1 and COX-2 in cancer cells were limited. In in vitro studies, Thoren and Jakobsson57 had demonstrated coordinate upregulation and downregulation of mPGES-1 and COX-2 in NSCLC cells. Jabbour and coworkers38 also found colocalization of mPGES-1 and COX-2 expression in neoplastic epithelial cells of endometrial carcinoma. Nonetheless, Yoshimatsu et al.26 observed marked differences in the extent of mPGES-1 and COX-2 expression in individual NSCLC tumors. In this study, we found no correlation between the expressions of mPGES-1 and COX-2, thus suggesting that the regulation of mPGES-1 and COX-2 in NSCLC might be different, but this requires further investigation and validation.

In this study, we observed an association between mPGES-1 overexpression and the patterns of tumor recurrence. A higher percentage of mPGES-1 over-expression was found in patients who developed lung metastases during follow-up, and the result was opposite for bony metastases. Although this finding is yet to be confirmed by further studies, the mechanism behind the organ preference in the process of metastasis is worth exploring. Several lines of evidence have indicated that mPGES-1 may be related to angiogenesis and tumor cell/environment interactions, in addition to the regulation of cell growth. Using PG receptor knockout mice, Amano et al.58 demonstrated the significance of PGE2 in promoting tumor angiogenesis. In autopsied NSCLC patients, upregulation of mPGES-1 was correlated with more tumor angiogenesis.59 Thus, mPGES-1 might regulate the metastatic pattern by modulating tumor angiogenesis.

Moreover, Paget,60 in 1889, proposed the "seed and soil" theory that metastasis depends on the interactions between selected cancer cells (seeds) and specific organ microenvironments (soil). Using gene transfection and cDNA array analyses, Kamei and colleagues41 showed that mPGES-1–directed cellular transformation was accompanied by changes in the expression of a variety of genes related to cyto-skeletal regulation and cell adhesion, including RhoA, ezrin, tubulin, annexins, and the adhesion molecules integrins and {alpha}1-catenin. The products of these genes are responsible for the regulation of actin filament rearrangement, assembly and stabilization of specialized plasma membrane domains, membrane fusion and focal adhesion, and so on.61–64 Hence, it is likely that through alternations in the expression of these genes, mPGES-1 may influence the site preference of the metastatic pattern. However, those deductions need further experimental verification.

In conclusion, our study demonstrated overexpression of mPGES-1 in human NSCLC, which was not associated with COX-2. No significant associations existed between the expression of mPGES-1 and clinicopathologic characteristics. Overexpression of mPGES-1 per se was not a significant prognostic indicator, but it might be implicated in the organ preference of metastasis. Further studies are needed to evaluate the potential of mPGES-1 as a target for cancer therapy.


   ACKNOWLEDGMENTS
 
The authors thank Kuan-Hua Chen, Chia-Li Lu and Li-Ling Yang for their excellent technical assistance. This work was supported by grants from the Taipei Veterans General Hospital (VGH93-64; Y-CW) and the Taiwan National Science Council (92-2314-B-039-009; C-TH).

Received for publication October 17, 2005. Accepted for publication January 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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