From the
Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan,
Department of Pathology, Toho University, School of Medicine, 5-21-16 Omori-Nishi, Ohta-ku, Tokyo 143-8540, Japan
Received for publication, December 31, 2002
, and in revised form, February 25, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
More direct evidence for the role of COX-2 and its product PGE2 in colorectal tumorigenesis has been provided by gene targeting studies. Gene disruption of either COX-2 (13) or the PGE receptor EP2 (14) results in reduction of the number and size of intestinal polyps in Apc mutant mice, a model for human familial adenomatous polyposis. In another model, disruption of the genes for the PGE receptors EP1 (15) or EP4 (16) suppresses the development of colorectal cancer induced by carcinogen. Moreover, gene knockout of cytosolic PLA2 (cPLA2
), which supplies the substrate arachidonic acid to COX-2, also leads to reduced polyposis in Apc mutant mice (17, 18).
PGES catalyzes the conversion of PGH2, which is produced from arachidonic acid by COX-1 or COX-2, to PGE2. Recent advances in this field have led to identification of at least three PGES enzymes, including cytosolic PGES (cPGES) (19), microsomal PGES (mPGES) -1 (20, 21, 22), and mPGES-2 (23). Among them, microsomal PGES-1 (mPGES-1) has received much attention, as this enzyme is induced by proinflammatory stimuli, down-regulated by anti-inflammatory glucocorticoids, and functionally coupled with COX-2 in marked preference to COX-1 (20, 21, 22). In comparison, cPGES (the heat shock protein-associated protein p23) is constitutively and ubiquitously expressed and is selectively coupled with COX-1 (19). mPGES-2 does not show homology with mPGES-1 and has a unique N-terminal hydrophobic domain and a glutaredoxin-like domain (23), although its cellular function has not yet been addressed.
mPGES-1 is a member of the MAPEG (for membrane-associated proteins involved in eicosanoid and glutathione metabolism) superfamily, to which other proteins involved in arachidonic acid metabolism, such as 5-lipoxygenase-activating protein (FLAP) and leukotriene C4 synthase, also belong (20, 21, 22). Induced expression of mPGES-1 has been postulated to be associated with various pathophysiological events in which COX-2-derived PGE2 has been implicated, such as rheumatoid arthritis (24), febrile response (25), reproduction (26, 27), bone metabolism (21), and Alzheimer's disease (28). A recent gene targeting study of mPGES-1 has shown that PGE2 production by lipopolysaccharide-stimulated peritoneal macrophages depends almost entirely on this enzyme (29). Induced expression of mPGES-1 is regulated by the NF-IL-6 pathway (29) or the mitogen-activated protein kinase pathway (30), the latter of which may switch on the inducible transcription factor Egr-1 that in turn binds to the proximal GC box in the mPGES-1 gene promoter, leading to mPGES-1 transcription (31).
A possible linkage of mPGES-1 with tumorigenesis has been provided by a recent observation that mPGES-1 is constitutively expressed in several cancers, most of which also express COX-2 constitutively (3234). In this study, we have used colon cancer cell lines and mPGES-1-transfected cells to examine the expression of mPGES-1 in colorectal cancer tissues and cells and evaluate its potential role in tumorigenesis.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of Antibody against Human mPGES-1Human mPGES-1 cDNA was subcloned into the bacterial expression vector pET21c (Novagen) and transformed into the competent cell BL21-D3 (Stratagene). After culture with 0.5 mM isopropyl--D-(-)-thiogalacto-pyranoside, cells were spun down, freeze-thawed, and suspended in phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 3 µg/ml leupeptin, 3 µg/ml antipain, 1 mM dithiothreitol, and 1% (v/v) sodium N-dodecanoylsalcosilate. After sonication and centrifugation for 10 min at 10,000 x g, the resultant supernatant was dialyzed against 20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl (TBS), 1 mM EDTA, and 0.5% (v/v) Triton X-100 overnight. Then the dialyzed sample was applied to a nickel-nitrilotriacetic acid-agarose column (Novagen), and the bound proteins were eluted with 4080 mM imidazole at a flow rate of 10 ml/h. Fractions containing pure His6-tagged mPGES-1 protein were collected and dialyzed against phosphate-buffered saline.
New Zealand White rabbits (male, 1 kg; Saitama Animal Center) were immunized subcutaneously with the purified mPGES-1 protein (0.3 mg/head) mixed with Freund's complete adjuvant (Difco). After several booster immunizations with Freund's incomplete adjuvant (Difco) at 2-week intervals, the blood was collected and the serum titer was assayed by enzyme-linked immunosorbent assay and Western blotting with recombinant mPGES-1 protein. The antiserum obtained was further purified on an immunoaffinity Hi-Trap NHS-activated column (Amersham Biosciences) that had been conjugated with mPGES-1 protein. The purified antibody was used in subsequent studies.
Transfection StudiesTransfection of cDNAs into HEK293 cells was performed by lipofection as described previously (21, 35). Briefly, 1 µg of plasmid (mPGES-1 in pCDNA3.1/hyg and COX-1 or -2 in pCDNA3.1/neo) was mixed with 2 µl of LipofectAMINE 2000 in 100 µl of Opti-MEM for 30 min and then added to cells that had attained 4060% confluence in 12-well plates (Iwaki Glass) containing 0.5 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 1 ml of fresh culture medium. After overnight culture, the medium was replaced with 1 ml of fresh medium and culture was continued at 37 °C in an incubator flushed with 5% CO2 in humidified air. The cells were cloned by limiting dilution in 96-well plates in culture medium containing appropriate antibiotics (10 µg/ml hygromycin or 1 mg/ml G418). After culture for 34 weeks, wells containing a single colony were chosen, and the expression of each protein was assessed by RNA blotting. The established clones were expanded and used for the experiments described below.
The C-terminal FLAG-tagged mPGES-1 cDNA was transfected into HCA-7 cells by the ViraPower lentiviral expression system (Invitrogen) according to the manufacturer's instructions. Briefly, the FLAG-tagged mPGES-1 cDNA insert was amplified by polymerase chain reaction with the Advantage cDNA polymerase mixture (Clontech) and was subcloned into the pLenyi6/V4-D-TOPO vector (Invitrogen). The resulting plasmid was transfected into 293FT cells (Invitrogen) with LipofectAMINE 2000, and an aliquot of the supernatant harvested 3 days after transfection was then added to HCA-7 cells. The cells were cultured in the presence of 40 µg/ml blastcidine (Invitrogen), and the antibiotic-resistant cells were used in subsequent studies.
Antisense ExperimentsHCA-7 cells (6 x 104 cells) were seeded into 6-well plates and cultured for 2 days. Then the mPGES-1 antisense S-oligonucleotide (0.2 nmol) 5'-GAGGAAGACCAGGAAGTGCAT-3' was transfected into HCA-7 cells with oligofectamine reagent. After 48 h, cell numbers and the PGE2 released into the supernatants during culture were quantified. The cell lysates were subjected to Western blotting to verify mPGES-1 expression.
Measurement of PGES ActivityPGES activity was measured by assessment of conversion of PGH2 to PGE2 as previously reported (21). Briefly, cells were harvested from culture dishes with a cell scraper and disrupted by sonication with a Branson sonifier (10 s, 3 times, 50% duty) in 10 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. After centrifugation of the sonicates at 100,000 x g for 1 h at 4 °C, the membrane fractions were used as an enzyme source. An aliquot (10 µg of protein equivalents) was incubated with 0.5 µg of PGH2 for 30 s at 24 °C in 0.1 ml of 0.1 M Tris-HCl (pH 8.0) containing 1 mM glutathione and 5 µg of indomethacin. After stopping the reaction by the addition of 100 mM FeCl2, PGE2 contents in the reactions were quantified by use of the enzyme immunoassay kit. Protein concentrations were determined by the bicinchoninic acid protein assay kit (Pierce) with bovine serum albumin as a standard.
RNA BlottingApproximately equal amounts (5 µg) of total RNA obtained from the cells were applied to separate lanes of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Biosciences) by random priming (Takara Biomedicals). All hybridizations were carried out as described previously (35).
SDS-PAGE/ImmunoblottingCell lysates (2 x 105 cell equivalents) were subjected to SDS-PAGE using 7.512.5% gels under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell) with a semi-dry blotter (MilliBlot-SDE system; Millipore). After blocking with 3% (w/v) skim milk in TBS containing 0.05% (v/v) Tween 20 (TBS-Tween), the membranes were probed with the respective antibodies (1:5,000 dilution for mPGES-1, cPGES, cPLA2, and COX-2, and 1:20,000 dilution for COX-1 and FLAG epitope in TBS-Tween) for 2 h, followed by incubation with horseradish peroxidase-conjugated anti-rabbit (for mPGES-1 and cPGES), anti-goat (for COXs), or anti-mouse (for FLAG) IgG (1:5,000 dilution in TBS-Tween) for 2 h, and were visualized with the ECL Western blot system (PerkinElmer Life Sciences) (35).
Semisoft Agar AssayCells (104 cells/ml) were suspended in cell culture medium containing 1% (w/v) low-melt agarose and plated on 60-mm culture dishes. After culture for 10 days at 37 °C in a CO2 incubator, colony numbers in each plate were counted. Relative colony size was determined by measuring 10 random colonies in each plate, and the mean for each treatment set was calculated and compared with that of controls.
Experiments with Nude MiceCells (5 x 106 cells) were suspended in 100 µl of phosphate-buffered saline and injected subcutaneously into BALB/c-nu/nu mice (6-week-old males) (Crea Japan). After 3 months, solid tumors were removed surgically and fixed in 10% (v/v) formalin. After embedding in paraffin, thin sections (46 µm thickness) of tumor tissues were prepared on glass slides.
ImmunohistochemistryThe tissue sections were incubated with Target Retrieval Solution (DAKO) as required, incubated for 10 min with 3% (v/v) H2O2, washed 3 times with TBS for 5 min each, incubated with 5% (v/v) skim milk for 30 min, washed 3 times with TBS-Tween for 5 min each, and incubated for 2 h with the first antibodies in TBS (1:50 and 1:200 dilutions for anti-mPGES-1 and anti-COX-2 antibodies, respectively). Then the sections were treated with the LSAB2 staining kit (for COX-2; DAKO) or the CSA system staining kit (for mPGES-1; DAKO).
cDNA Array AnalysismRNAs isolated from COX-2-expressing and COX-2/mPGES-1-coexpressing HEK293 cells (107 cells for each) were reverse-transcribed into cDNA and 32P-labeled with Atlas cDNA Expression Arrays kit (Clontech). Hybridization was performed on the Atlas Human 1.2 Array (Clontech). After exposure to an image plate (BAS III; Fuji Photo Film), signals were analyzed by AtlasImage 1.0 Software (Clontech).
Other ProceduresConfocal laser microscopy was performed as described previously (21). Data were analyzed by Student's t test. Results are expressed as the mean ± S.E., with p = 0.05 as the limit of significance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
mPGES-1 Is Involved in the Growth of Human Colon Adenocarcinoma Cell Line HCA-7Fig. 2A depicts the expression of the PGE2-biosynthetic enzymes cPLA2, COX-1, and -2, cPGES and mPGES-1 in 3 human colon cancer cell lines (HCA-7, WiDr, and HCT116) as well as in HEK293 cells. Of these cell lines, only HCA-7, a colon adenocarcinoma cell line that has been reported to exhibit COX-2- and PGE2-dependent growth (4, 36, 37), expressed COX-2 and mPGES-1 constitutively. mPGES-1 expression was also detected in WiDr and HCT116 cells, although rather more weakly than in HCA-7 cells. Indirect immunofluorescent cytostaining analysis by confocal microscopy revealed the colocalization of COX-2 and mPGES-1 in the perinuclear area (Fig. 2B). All these cell lines expressed cPLA2
and cPGES constitutively, whereas COX-1 expression was restricted to HCA-7 and WiDr cells (Fig. 2A). Stimulation of HCA-7 cells with IL-1
, a proinflammatory cytokine that increases the expression of COX-2 and mPGES-1 in various cell types (20, 21, 22, 24, 25), did not alter the expression of COX-2 (not shown) and mPGES-1 (Fig. 2C). We therefore chose HCA-7 cells as a model for further study.
|
To examine the role of PGE2 produced by the COX-2/mPGES-1 pathway in the growth of HCA-7 cells, we first examined the effects of NS-398, a well known COX-2 inhibitor (38), and MK-886, an mPGES-1 inhibitor that also inhibits other MAPEG proteins such as FLAP and LTC4 synthase (22), on cell growth and PGE2 production. Treatment of HCA-7 cells with NS-398 almost completely abolished PGE2 production, accompanied by 40% reduction of cell growth (Fig. 2D). When HCA-7 cells were treated with MK-886 at a concentration completely inhibiting mPGES-1 enzymatic activity in vitro (data not shown), there was a
40% reduction in cell growth (as in the case of NS-398 treatment), even though the inhibition of PGE2 production was only partial (
60%) (Fig. 2E).
To assess the contribution of mPGES-1 to cell growth and PGE2 production in a more comprehensive way, we used an antisense oligonucleotide for mPGES-1 to reduce its expression. Treating the cells with an mPGES-1-specific antisense oligonucleotide resulted in marked reduction of mPGES-1 protein expression with no appreciable change in COX-2 expression (Fig. 3A). In this setting, both cell growth (Fig. 3B) and PGE2 production (Fig. 3C) were reduced partially, as observed in the experiments with MK-886 (Fig. 2E). The production of PGF2 was unaffected by antisense treatment (Fig. 3D), verifying that the antisense acted specifically on mPGES-1 but not on upstream enzymes or other terminal enzymes. Control oligonucleotide did not affect mPGES-1 expression, PGE2 production, or cell growth (not shown). These results suggest that PGE2 produced via the COX-2/mPGES-1 pathway is partially involved in the proliferation of HCA-7 cells.
|
We next examined if, conversely, overexpression of mPGES-1 would facilitate the growth of HCA-7 cells. To this end, C-terminal FLAG-tagged mPGES-1 cDNA was transfected into HCA-7 cells by lentivirus-mediated gene transfer. As shown in Fig. 4A, the virus-infected cells expressed FLAG-tagged mPGES-1 protein just above the position of endogenous mPGES-1, whereas the constitutive expression of the upstream enzymes cPLA2 and COX-2 was unaltered. PGES activity in vitro in the membrane fraction of cell lysates (Fig. 4B) and PGE2 release into medium during culture (Fig. 4C) were markedly increased in mPGES-1-transfected cells relative to control cells. Furthermore, the cell growth rate of mPGES-1-transfected cells was significantly faster than that of control cells (Fig. 4D).
|
Transformation of HEK293 Cells by Overexpression of mPGES-1As shown in Fig. 5A, HEK293 cells cotransfected with COX-2 and mPGES-1 grew more rapidly than parental cells over the entire culture periods. COX-2/mPGES-1 cotransfection into HEK293 cells led to cell aggregation, rounding and piling up, and both enzymes were colocalized in the perinuclear region in the aggregated cells (Fig. 5B). These morphological changes were less pronounced in replicate COX-2/mPGES-1-cotransfected cells cultured in the continued presence of NS-398, added immediately after transfection, or in cells cotransfected with COX-2 and mPGES-1-R110S, which has very weak enzyme activity (21) (Fig. 5B). These results suggest that the catalytic functions of COX-2 and mPGES-1 are both required for triggering cellular transformation. However, addition of NS-398 to already transformed COX-2/mPGES-1-expressing clones failed to reverse their growth and aggregated morphology (not shown), indicating that transformation of HEK293 cells by COX-2/mPGES-1 cotransfection is an irreversible event.
|
As anchorage-independent growth is considered to be an in vitro test for tumorigenesis, we examined the growth of COX-2/mPGES-1-cotransfected HEK293 cells in a semisoft agar medium. As demonstrated in Fig. 6A, the COX-2/mPGES-1-coexpressing cells exhibited marked anchorage-independent growth, as manifested by the appearance of a number of large colonies. On the other hand, cells expressing COX-2 alone or cells coexpressing COX-1 and mPGES-1 formed fewer and smaller colonies, and parental cells did not grow appreciably in soft agar (Fig. 6A). Quantification of the numbers and sizes of colonies formed in this colony assay is summarized in Fig. 6B.
|
When parental and COX-2/mPGES-1-expressing HEK293 cells, as well as HCA-7 cells used as a positive control, were injected subcutaneously into athymic nude mice, COX-2/mPGES-1-expressing HEK293 cells and HCA-7 cells, but not parental HEK293 cells, formed large solid tumors after 3 months (Fig. 7A). Histopathologic examination of a fraction of whole tumor tissues from COX-2/mPGES-1-expressing HEK293 cell xenografts is shown in Fig. 7B. At the site of implantation, a whitish nodular tumor was formed in the subcutaneous tissue and exhibited a well circumscribed mass (Fig. 7B, panel a). The tumor cells had swollen nuclei of round or polygonal shape with sporadic mitosis, and their cytoplasm was scare and chromophobic (Fig. 7B, panel b). These characteristic features of the tumor suggest its malignancy. The tumor cells were proliferating with scanty interstitium that was mainly composed of capillaries (Fig. 7B, panel c, arrow). These capillaries consisted of swollen endothelial cells, and were likely to be newly formed vessels. Immunohistochemistry for mPGES-1 (Fig. 7B, panel d) and COX-2 (Fig. 7B, panel e) revealed the location of both enzymes around the nuclei in tumor cells.
|
The HCA-7 xenograft tumor appeared as a nodular mass and was well demarcated (Fig. 7C, panel a). Tumor with a thin fibrous capsule was visible in the subcutaneous region (Fig. 7C, panel b, arrow). The tumor cells had scare cytoplasm and hyperchromatic nuclei of various sizes with sporadic mitosis, and were partially proliferating with tubular formation. These features indicated adenocarcinoma differentiation (Fig. 7C, panel c). Immunoreactivities for mPGES-1 (Fig. 7C, panel d) and COX-2 (Fig. 7C, panel e) were detected in the tumor regions.
Identification of a Panel of Genes Altered by Overexpression of mPGES-1To further elucidate the mechanisms of cellular transformation by co-overexpression of COX-2/mPGES-1, we surveyed a panel of genes, the expression of which was significantly altered in COX-2/mPGES-1-cotransfected HEK293 cells relative to cells expressing COX-2 alone, by cDNA array analysis. As summarized in Table I, increased genes included those required for cytoskeletal regulation (the small G protein RhoA), cell growth (the receptor tyrosine kinases ErbB3 and Flt1, the cyclin-dependent kinase CDK5, and the tumor necrosis factor signaling molecule TRAF-1), gene transcription (c-Myc, GATA4, and YL-1), protein synthesis (the ribosomal proteins S19 and S3A), and so on. Conversely, decreased genes included the subunit of protein phosphatase 1, cytoskeleton regulators (tubulin
and ezrin), cell adhesion regulators (several integrins and
1-catenin), the transcription factor Egr-1, and thymosins (Table I).
|
We then performed Northern and Western blot analyses to verify that the expression of these genes identified by cDNA array indeed differ between COX-2- and COX-2/mPGES-1-transfected HEK293 cells and between parental and mPGES-1-transfected HCA-7 cells. A representative result of the blotting analyses is shown in Fig. 8. In line with the cDNA array analysis, the expression levels of rhoA, c-myc, and ErbB3 were markedly increased, whereas those of ezrin and Egr-1 were decreased, in COX-2/mPGES-1-cotransfected cells relative to COX-2-transfected cells (Fig. 8A). Moreover, increased expression of rhoA, c-myc, and ErbB3 was also observed in mPGES-1-transfected HCA-7 cells as compared with parental cells (Fig. 8B).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Given these observations, the aim of this study was to evaluate the potential contribution of mPGES-1, which lies downstream of the COX-2-dependent PGE2-biosynthetic pathway, to tumorigenesis. Elevated expression of mPGES-1 in human cancers has recently been demonstrated in non-small cell lung cancer (32) and endometrial adenocarcinoma (33). In this study, we performed immunohistochemistry of human colon cancer and adenoma tissues with anti-COX-2 and anti-mPGES-1 antibodies and found that both enzymes are coexpressed in many, even if not all, of malignant and benign colorectal tumor cells (Fig. 1). While this study was underway, Yoshimatsu et al. (34) reported that mPGES-1 is overexpressed in >80% of human colorectal tumors and adenomas, in line with our present observation.
To assess the role of mPGES-1 in growth of colon cancer cells in cell culture, we took advantage of HCA-7 cells, a human colon cancer cell line that has often been used to investigate COX-2-dependent tumorigenesis (4, 36, 37). Proliferation of HCA-7 cells is reduced by COX-2 inhibitors, and this is reversed by exogenous PGE2 (4, 36). PGE2 also prevents COX-2 inhibitor-induced apoptosis by inducing expression of Bcl-2, and increases clonogenicity in HCA-7 cells (37). In this study, we found that both mPGES-1 and COX-2 are constitutively, but not inducibly, expressed in HCA-7 cells (Fig. 2A). Although the transcriptional regulations of COX-2 and mPGES-1 are not entirely identical (24, 31), recent observations that the expression of COX-2 in HCA-7 cells is transcriptionally regulated by the NF-IL-6-regulatory element (41) and that mPGES-1 induction in lipopolysaccharide-stimulated mouse macrophages is ablated in NF-IL-6-deficient mice (29) suggest a role for this transcription factor in the expression of both COX-2 and mPGES-1 in HCA-7 cells.
Importantly, studies using MK-886, an inhibitor for MAPEG proteins including mPGES-1 (Fig. 2, C and D), an mPGES-1-specific antisense oligonucleotide (Fig. 3) and overexpression of mPGES-1 (Fig. 4) together suggest that mPGES-1 is involved in PGE2 production and proliferation of HCA-7 cells, even if partially. Somewhat paradoxically, under conditions where the COX-2 inhibitor NS-398 almost completely abolished PGE2 production, reduction of cell growth by NS-398 was still only partial, whereas reduction of PGE2 production and cell growth by MK-886 or the mPGES-1 antisense was partial and parallel (Figs. 2 and 3). A likely explanation for these observations is that the PGE2 produced via the COX-2/mPGES-1 pathway contributes to cell growth, whereas there is an additional COX-2-dependent but mPGES-1-independent PGE2-biosynthetic route that is unrelated to the mitogenic response. Polarized, vectorial production of PGE2 toward basolateral and apical directions in HCA-7 cells (36) may support the existence of distinct PGE2 pools. Considering that the growth-stimulating effect of PGE2 on HCA-7 cells depends, at least in part, on EP4 (16), the PGE2 produced by COX-2/mPGES-1 in the perinuclear region may be preferentially presented to this G-protein-coupled PGE2 receptor through an unknown mechanism. Alternatively, the perinuclear PGE2 (or possibly other unknown metabolites) produced by COX-2/mPGES-1 may act on certain nuclear receptors that in turn promote cell growth. Indeed, several eicosanoids have been shown to stimulate the peroxi-some proliferator-activated receptor family of nuclear receptors (43, 44). Nonetheless, different sensitivity of PGE2 production by HCA-7 cells to COX-2 and mPGES-1 inhibitors and mPGES-1 antisense oligonucleotide strongly argues that this cell line may contain an alternative COX-2-dependent PGE2-biosynthetic route that involves other PGES enzyme(s). Although cPGES is abundantly expressed in HCA-7 cells (Fig. 2A), this enzyme has been reported to be coupled rather specifically with COX-1, not COX-2 (19). mPGES-2, a recently identified enzyme whose transcript is detected in human colon (22), may represent a second PGES that can be coupled with COX-2, a possibility that is now under investigation.
In another model using HEK293 cells, cotransfection of COX-2 and mPGES-1 led to cellular transformation, as demonstrated by rapid proliferation, morphological change, piling up, and aggregation in normal culture, large colony formation in soft agar culture, and formation of solid tumors in nude mice (Figs. 5, 6, 7). Immunohistochemical examination of the tumor revealed tissue invasion by COX-2/mPGES-1-derived tumor cells as well as angiogenesis (Fig. 7), consistent with the angiogenic effect of COX-2 and PGE2 (11, 45, 46). Growth promotion and morphological change of COX-2/mPGES-1-transfected HEK293 cells in culture were less pronounced if NS-398 was added immediately after COX-2/mPGES-1 transfection, or if a catalytically inactive mPGES-1 mutant was transfected in place of native enzyme (Fig. 5C). Once transformed, however, neither NS-398 nor MK-886 reversed the growth and aberrant morphology of COX-2/mPGES-1-cotransfected HEK293 cells. These observations indicate that, even though the COX-2/mPGES-1 catalytic product triggers cellular transformation, it may not be required for subsequent maintenance of a transformed phenotype in this setting. A similar event has been observed in ECV endothelial cells transfected with COX-1 (not COX-2, which induced apoptosis in these cells), where aggressive growth of COX-1-transfected ECV cells was no longer inhibited by indomethacin (47). Unlike HCA-7 cells (4, 36), simple addition of exogenous PGE2 to HEK293 cells did not induce cellular transformation, as mentioned previously (21). The following possibilities could be considered: (i) continuous production of high levels of PGE2 around the perinuclear area by COX-2/mPGES-1 is critical for inducing transformation; (ii) some additional components, which act cooperatively with PGE2 to induce cellular transformation, are induced by COX-2/mPGES-1 overexpression; and (iii) some unknown substances produced by COX-2/mPGES-1 may be involved in transformation.
To gain insights into mPGES-1-promoted cellular transformation, we sought to identify mPGES-1-regulated genes by cDNA array technology (Table I and Fig. 8). The genes identified so far can be categorized into several groups: (i) genes for signaling molecules related to cell proliferation and differentiation; (ii) genes for transcription factors; (iii) genes related to cytoskeletal regulation; (iv) cell adhesion molecules; and (v) genes with unknown functions. Some of the induced genes are proto-oncogenes that have the capacity to promote cellular transformation when transfected alone into cells.
Increased genes related to cell growth and differentiation include those encoding the receptor tyrosine kinases ErbB3 and Flt1, the cyclin-dependent protein kinase CDK5, the ribosomal proteins S3A and S19, the proliferation-associated nucleolar protein NOL1, and TRAF1. ErbB3, a ligand for heregulin, is a member of the epidermal growth factor receptor family and its overexpression has been frequently found in human tumors (48). Induced expression of ErbB3 is in line with a recent report that PGE2 transactivates epidermal growth factor receptor, thereby switching on the mitogenic signaling pathway in gastric epithelial and colon cancer cell lines (49). Moreover, COX-2 is overexpressed in hereglin 2-positive breast cancer (50). Induction of Flt1, a receptor for vascular endothelial cell growth factor (51, 52), may be linked to angiogenesis, which has been associated with COX-2- and PGE2-dependent tumor development (11, 45, 46).
Cyclin-dependent protein kinases generally play crucial roles in cell cycle progression (53). Apart from cell cycle control, CDK5 phosphorylates a diverse list of substrates and regulates a range of cellular processes, including cell adhesion and motility (54, 55). Ribosomal proteins are integral components of the basal cellular machinery involved in protein synthesis and have been found to play roles in regulating cell growth and transformation (56, 57). NOL1 expression is associated with cell proliferation during G1-S phases and represents a biological marker indicative of tumor aggressiveness, particularly the late events of colorectal tumor progression (58, 59). TRAF1 is a signal transducer of the tumor necrosis factor receptor family, which has been implicated in cell differentiation (60). Conversely, decreased expression of protein phosphatase 1, a tumor suppressor of which mutations have been found in lymphoma and hepatoma (61, 62), may also account for aggressive cell growth of COX-2/mPGES-1 cotransfectants.
Transcription factors whose expressions are altered by COX-2/mPGES-1 include c-Myc, GATA4, and YL-1 (increased) as well as Egr-1 (decreased). Induced expression of c-Myc, a helix-loop-helix transcription factor, is of great importance in controlling cell growth and vitality (63), and is commonly amplified in many human tumors (64). Involvement of c-Myc in colon cancer development downstream of the Wnt/APC/-catenin signaling pathway has been documented (65, 66). Moreover, the activation of the Wnt/APC/
-catenin signaling pathway results in transcriptional up-regulation of COX-2 in cancer cells (67). GATA-4, a member of the GATA transcription factor family, plays a role in the regulation of cell migration and its overexpression has been found in several types of cancer (68). YL-1, also reported as transcription factor-like 1, has been implicated in anchorage-independent cell growth (69). Egr-1, a COX-2/mPGES-1-decreased gene, is an inducible transcription factor that binds to GC-rich elements and plays a crucial role in transcriptional activation of the mPGES-1 gene (31). Hence, the reduction of Egr-1 expression in COX-2/mPGES-1 cotransfectants may be a reflection of negative feedback regulation of mPGES-1 expression.
Altered genes encoding proteins for cytoskeletal regulation include RhoA (increased), ezrin, tubulin, and annexins (decreased), which may be responsible for the marked morphological change in COX-2/mPGES-1 cotransfectants. The small G protein RhoA regulates various aspects of actin filament rearrangement and has a key role in growth of tumors (70, 71). Ezrin plays structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains, particularly in surface projections such as microvilli and membrane ruffles where it links the microfilaments to the plasma membrane (72). Annexins interact with cytoskeletal proteins and have been implicated in diverse cellular responses including differentiation and membrane fusion (73). Because annexins are capable of inhibiting cPLA2 (74), decreased expression of annexins may lead to increased activation of cPLA2
, thereby amplifying the COX-2/mPGES-1-dependent PGE2 biosynthesis. Indeed, cPLA2
has often been associated with cellular transformation (75) and cPLA2
knockout decreases the incidence of polyposis in Apc mutant mice (17, 18). There were decreases in the expression of several cell adhesion molecules, such as integrins and
1-catenin, which are critical components for focal adhesion (76, 77). Thus, decreased expression of these focal adhesion proteins is consistent with the reduced anchorage dependence of COX-2/mPGES-1 cotransfectants.
The most remarkably decreased genes in COX-2/mPGES-1 cotransfectants are those for thymosins. Prothymosin is a small highly acidic protein found in the nuclei of virtually all mammalian tissues, and its high conservation in mammals and wide tissue distribution suggest an essential biological role (42). Although the exact mechanism of action of thymosins remains elusive, the one constant has been their relationship with the proliferating state of the cell and its requirement for cellular growth and survival. Overall, although the mechanisms by which overexpression of COX-2/mPGES-1 alters the expression of these genes, and whether these genes, alone or in combination, induce transformation of HEK293 cells, are currently unclear, the present findings shed light on unexplored aspects of the combined action of COX-2 and mPGES-1 in tumorigenesis. Indeed, increases in some of the genes identified in COX-2/mPGES-1-transfected HEK293 cells were also observed in mPGES-1-transfected HCA-7 cells (Fig. 8B), suggesting that our findings could be applicable to at least some types of colorectal cancer. Evaluation of the effect of COX-2/mPGES in non-transformed intestinal cell lines will give further insight into this critical issue.
In summary, this study demonstrates that mPGES-1, in concert with COX-2, can be associated with cellular transformation and cancer development. Future studies using mPGES-1 knockout mice or mPGES-1-specific inhibitors would open further insights into the role of this critical PGE2 biosynthetic terminal enzyme in tumorigenesis. Importantly, our observation that mPGES-1 is overexpressed in colorectal tumors provides the basis for future studies that will evaluate whether mPGES-1 is a bona fide therapeutic target.
![]() |
FOOTNOTES |
---|
¶ To whom correspondence should be addressed. Tel.: 81-3-3784-8196; Fax: 81-3-3784-8245; E-mail: kudo{at}pharm.showa-u.ac.jp.
1 The abbreviations used are: PG, prostaglandin; COX, cyclooxygenase; PLA2, phospholipase A2; mPGES-1, microsomal prostaglandin E2 synthase-1; MAPEG, membrane-associated proteins involved in eicosanoid and glutathione metabolism; TBS, Tris-buffered saline; IL, interleukin; HEK, human embryonic kidney.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|