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Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The University of Edinburgh Academic Center, Edinburgh, Scotland EH16 4SB, United Kingdom
Address all correspondence and requests for reprints to: Dr. Henry N. Jabbour, Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, The University of Edinburgh Academic Center, 49 Little France Crescent, Old Dalkeith Road, Edinburgh, Scotland EH16 4SB, United Kingdom. E-mail: h.jabbour{at}hrsu.mrc.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PGs are biosynthesized respectively by COX enzymes and specific terminal prostanoid synthase enzymes. Currently, there are three known isoforms of COX enzyme, COX-1, COX-2, and COX-3 (a splice variant of COX-1) (3, 8). Most tumors that highly express COX enzymes, including reproductive tract cancers such as endometrial adenocarcinoma and cervical carcinoma, have also been found to contain high levels of PGE2 and PGE synthase (3, 9, 10). PGE2 exerts its autocrine/paracrine effects on target cells and tissues by coupling to four subtypes of G protein-coupled receptors (GPCRs), which have been pharmacologically classified as EP1, EP2, EP3, and EP4 (E-series prostanoid receptors) (11). These receptors are often coexpressed together in the same cell and use alternate and in some cases opposing intracellular signaling pathways (12). PGE2 interaction with EP1 receptor mobilizes inositol trisphosphates and intracellular calcium via Gq. EP2 and EP4 receptors couple to G
s and activate adenylyl cyclase, resulting in increased formation of cAMP. Several splice variants have been reported for the EP3 receptor; these are coupled to different signaling pathways resulting in Gs, Gi, or Gq signaling depending on the specific splice variant and cell type (13, 14, 15). In reproductive cancers, such as endometrial and cervical carcinoma, the expression and the Gs signaling of the EP2 and EP4 receptor are elevated (9, 10), suggesting an autocrine/paracrine regulation of endometrial tumor function by PGE2. Similar observations correlating PGE2-EP receptor interaction and tumor function have been made in other model systems (5, 13, 16, 17, 18, 19, 20, 21, 22). In APC
716 mice, a murine model for familial adenomatous polyposis, enhanced angiogenic potential and microvascular density in intestinal polyps correlates strongly with COX-2, EP2 receptor and angiogenic factor expression (19). Ablation of the EP2 receptor in these mice results in a decrease in the size of intestinal polyps coincident with a decrease in COX-2 and angiogenic factor expression (20). Similarly, in human pancreatic cancer cells, PGE2 produced by COX-2 increases vascular endothelial growth factor (VEGF) expression via the EP2 receptor (18). These studies therefore show that the PGE2-EP2 receptor interaction may promote tumorigenesis by promoting neovascularization and angiogenesis. However, the intracellular pathways associated with EP2 receptor signaling to angiogenic factors such as VEGF are not elucidated.
Other EP receptor subtypes, either coexpressed in the same cell or on adjacent cells, may act synergistically to promote tumorigenesis by targeting signal transduction cascades and genes involved in growth and proliferation. For example, in colorectal carcinoma cells, PGE2 interacts with the EP4 receptor to promote cellular growth, migration and invasiveness (5). This effect of PGE2 stimulation of the EP4 receptor is shown to involve productive cross-communication between the EP4 receptor and the epidermal growth factor receptor (EGFR)-protein kinase B/Akt signaling pathways (5, 17). Further evidence in support of a role for the EP4 receptor in tumor growth has been shown recently by Fujino et al. (22), where PGE2, via the EP4 receptor, can induce the expression of early growth response factor via the protein kinase B/Akt and ERK1/2 signaling pathways.
In this study, we investigated the potential role of elevated EP2 receptor expression and signaling in modulating vascular function in endometrial adenocarcinomas, by investigating the molecular signal transduction pathways associated with VEGF expression via the EP2 receptor using an endometrial adenocarcinoma (Ishikawa) cell model system. We found that treatment of EP2 receptor overexpressing, compared with wild-type and EP2 antisense (AS) Ishikawa cells, with PGE2 rapidly augments the activation of the EGFR and ERK1/2 signaling pathways in a predominantly intracellular cAMP-dependent protein kinase A (PKA)- and c-Src-mediated manner, resulting in an increase in the mRNA expression and secretion of VEGF. We also show that inhibition of EGFR function by chemical inhibition and RNA interference blocks the induction and secretion of VEGF, indicating that the EGFR is necessary for transducing signaling via the EP2 receptor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) performed on cellular extracts from wild-type, EP2 S, and EP2 AS cells confirmed elevated EP2 receptor expression in Ishikawa EP2 S cells and reduced expression of EP2 receptor in AS cells compared with wild-type cells. The expression of EP2 receptor in Ishikawa S cells was determined to be 4.2 ± 1.8- and 9.8 ± 0.5-fold greater in Ishikawa EP2 S cells compared with wild-type and EP2 AS cells respectively (P < 0.01). Expression of EP2 receptor in AS cells was determined to be 2.3 ± 0.8-fold less compared with wild-type cells (P < 0.05). Immunofluorescence microscopy showed elevated EP2 receptor immunoreactivity in EP2 S cells and reduced EP2 immunoreactivity in AS cells compared with wild-type cells. EP2 expression was localized to the plasma membrane (Fig. 1B). Incubating cells with rabbit IgG abolished the immunoreactivity (Fig. 1B, control representative of EP2 receptor S cells; C). Previously, we reported elevated EP2 receptor expression in endometrial adenocarcinomas (9). To determine how our Ishikawa EP2 receptor model system compares with the EP2 receptor expression detected in endometrial adenocarcinomas in vivo, we performed real-time quantitative RT-PCR analysis on endometrial adenocarcinoma tissues and Ishikawa wild-type and EP2 S cells. As shown in Fig. 1C, the relative EP2 receptor mRNA expression in EP2 S cells and adenocarcinoma tissues was significantly greater than the EP2 receptor expression in wild-type Ishikawa cells (P < 0.05). EP2 receptor couples to Gs and activate adenylyl cyclase resulting in increased formation of cAMP. We measured intracellular cAMP accumulation in wild-type, EP2 S and EP2 AS cells in response to administration of 100 nM PGE2. A time-dependent increase in cAMP accumulation was observed in wild-type, EP2 S, and EP2 AS cells after treatment with PGE2. This agonist-induced cAMP accumulation was significantly elevated in EP2 S cells compared with wild-type and EP2 AS cells (P < 0.05). Accumulation of cAMP was significantly reduced in EP2 AS cells compared with wild-type cells (P < 0.05). Moreover, the elevation in cAMP accumulation in EP2 S cells treated with PGE2 compared with wild-type cells was similar to previous observations of cAMP generated in endometrial adenocarcinoma explants in response to PGE2 (3.2 ± 0.3 vs. 3.4 ± 0.4; P < 0.05; Ref.9).
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, lanes 1 and 2; P < 0.05) stimulated with PGE2. Treatment of cells with inhibitors of PKA (H-89, lane 3), c-Src (PP2, lane 4) and EGFR kinase (AG1478, lane 6) abolished the PGE2-induced EGFR tyrosine phosphorylation (P < 0.05). Treatment of the cells with a general matrix metalloproteinase inhibitor (MMP, GM6001, lane 5) attenuated the PGE2-induced phosphorylation of the EGFR; however, the reduction was not statistically significant.
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, lane 2, P < 0.05) in response to treatment with dibutyryl cAMP compared with cells stimulated with vehicle alone (Fig. 3, lane 1). Treatment of cells with inhibitors of PKA (H-89, Fig. 3, lane 3), Src (PP2, Fig. 3, lane 4) and EGFR kinase (AG1478, Fig. 3, lane 5) abolished this action of dibutyryl cAMP. In parallel, Ishikawa EP2 S cells were treated with 10 nM EGF for 10 min in the presence or absence of H-89, PP2, and AG1478. EGF induced a profound tyrosine phosphorylation of the EGFR (Fig. 3, lane 6). This phosphorylation was not inhibited by cotreatment with H-89 (Fig. 3, lane 7), or PP2 (Fig. 3, lane 8) and abolished by the AG1478 (Fig. 3, lane 9).
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). The ERK1/2 activation was significantly elevated in Ishikawa EP2 S cells (Fig. 4, panel 2) compared with wild-type and EP2 AS cells. The greatest ERK1/2 activation was seen in Ishikawa EP2 S cells, after treatment with 100 nM PGE2 for 1 min (Fig. 4, lane 2), compared with cells treated with vehicle alone (Fig. 4, lane 1). No significant phosphorylation of p38 or JNK MAPK was observed in wild-type, S, or AS cells after treatment of cells with 100 nM PGE2 within the 10-min time frame (data not shown).
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, ERK1/2 phosphorylation was most dramatically elevated in EP2 S cells, compared with wild-type and EP2 AS (Fig. 5A, lane 2). This elevation in ERK1/2 activation was abolished by treatment of Ishikawa cells with H-89 (Fig. 5A lane 3), PP2 (Fig. 5A lane 4), and AG1478 (Fig. 5A, lane 6). Treatment of cells with the MMP inhibitor (GM6001) attenuated the PGE2-induced ERK1/2 phosphorylation; however, the reduction was not statistically significant (Fig. 5A, lane 5).
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, lane 2) after treatment with dibutyryl cAMP compared with cells stimulated with vehicle alone (Fig. 5B, lane 1). Preincubation of cells with H-89 (Fig. 5B, lane 3), PP2 (Fig. 5B, lane 4), and AG1478 (Fig. 5B, lane 5) abolished this action of dibutyryl cAMP. In parallel, we treated Ishikawa S cells with 10 nM EGF for 10 min in the presence or absence of the same panel of chemical inhibitors. EGF-induced ERK1/2 activation (Fig. 5B, lane 6), was insensitive to cotreatment with H-89 (Fig. 5B, lane 7), or PP2 (Fig. 5B, lane 8) but was almost completely inhibited with AG1478 (Fig. 5B, lane 9).
EP2 Receptor Activation Induces VEGF Expression and Secretion via Transactivation of EGFR
The role of PGE2-EP2 receptor signaling on the expression of VEGF was investigated by quantitative RT-PCR analysis after stimulation of wild-type, EP2 S, and EP2 AS Ishikawa cells with 100 nM PGE2 or vehicle (48 h). As shown in Fig. 6A, PGE2 stimulation resulted in a 3.5 ± 1.1-fold increase in the expression of VEGF in Ishikawa EP2 S cells (P < 0.01). However, no such increase in the expression of VEGF was observed in wild-type or EP2 AS cells in response to PGE2 treatment. Treatment of cells with AG1478 (P < 0.05) and the inhibitor of MAPK kinase (MEK1/2, PD98059, P < 0.01) significantly reduced the PGE2-induced expression of VEGF. Interestingly, treatment of EP2 S cells with dibutyryl cAMP could also increase the expression of VEGF in a dose-dependent manner, with 5 mM dibutyryl cAMP generating the most robust signal (2.5 ± 0.1-fold increase in the expression of VEGF; data not presented) after 48 h. This cAMP-mediated elevation in VEGF expression was inhibited by cotreatment of cells with AG1478 and PD98059. Incubation of cells with the specific inhibitors on their own did not result in any significant alteration in mRNA levels over the 48-h time period at the concentrations used compared with cells treated with vehicle alone (data not shown). To further implicate PGE2-mediated EGFR transactivation in modulation of the expression of VEGF, Ishikawa EP2 S cells were transfected with small inhibitory RNA (siRNA) oligonucleotides targeted against the EGFR, or control random siRNA oligonucleotides and treated with 100 nM PGE2 for 48 h. As shown in Fig. 6B, transfection of Ishikawa EP2 S cells with EGFR siRNA completely abolished the PGE2-induced expression of VEGF, compared with cells transfected with the random control siRNA (P < 0.01). Western blot analysis for EGFR expression in Ishikawa EP2 S cells transfected with EGFR siRNA confirmed a reduction in EGFR protein compared with cells transfected with control siRNA (Fig. 6C).
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, PGE2 treatment of EP2 S cells induced a significant elevation in VEGF protein in the culture medium (P < 0.01). No significant elevation in secreted VEGF protein was observed in wild-type and EP2 AS cells after treatment with PGE2. The cotreatment of cells with AG1478 (P < 0.01) and PD98059 (P < 0.01) abolished the PGE2-induced secretion of VEGF into the culture medium. Similarly, transfection of Ishikawa EP2 S cells with EGFR siRNA for 48 h abolished the elevated VEGF protein secreted into the culture medium, compared with EP2 S cells transfected with control random siRNA (Fig. 7B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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716 mice, enhanced angiogenic potential and microvascular density in intestinal polyps correlates strongly with EP2 receptor and angiogenic factor expression (19). Ablation of the EP2 receptor in these mice results in a decrease in the size of intestinal polyps coincident with a decrease in angiogenic factor expression (20). In human pancreatic cancer cells, PGE2 produced by COX-2 increases VEGF expression via the EP2 receptor (18). In addition to modulation of angiogenic factor expression by the COX/PGE2 biosynthetic pathway, overexpression of COX-2 and subsequent elevated synthesis of PGE2 in Ishikawa cells can also induce the expression of EP2 receptor and attenuate the expression of antiangiogenic factors (30). The molecular signal transduction pathways mediating the effects of PGE2 and its cognate receptors in endometrial pathologies remains to be elucidated. To our knowledge, this is the first study to investigate EP2 receptor signaling in endometrial adenocarcinoma cells. Previous findings in our laboratory demonstrated elevated expression and cAMP signaling of EP2 receptor in endometrial adenocarcinomas, localized in neoplastic epithelial and endothelial cells suggesting an autocrine/paracrine regulation of tumorigenesis by PGE2 via the EP2 receptor (9). To elucidate the intracellular signal transduction pathways mediating the effects of PGE2 via the EP2 receptor in endometrial adenocarcinomas, we established an endometrial adenocarcinoma cell model system using Ishikawa human endometrial adenocarcinoma cells. For this purpose, we artificially overexpressed EP2 receptor in Ishikawa cells by introducing the EP2 receptor cDNA in the S and AS orientation. This was done to elevate the levels of EP2 receptor in the Ishikawa cell line to the levels observed in endometrial adenocarcinomas to create a model system which best represents EP2 signaling in endometrial adenocarcinoma in vivo. EP2 receptor expression in Ishikawa wild-type, EP2 S and EP2 AS cells was confirmed by Western blot analysis, immunofluorescence microscopy, and accumulation of intracellular cAMP. Elevated EP2 receptor expression was observed in Ishikawa EP2 S cells, compared with wild-type and EP2 AS cells and localized to the plasma membrane compartment. The expression of EP2 receptor in EP2 S cells also correlated closely with expression levels of EP2 receptor in endometrial adenocarcinomas. This elevation in EP2 receptor expression was associated with an elevation in accumulation of intracellular cAMP in Ishikawa EP2 S cells, and a reduction in cAMP accumulation in EP2 AS cells compared with wild-type Ishikawa cells. Moreover, the cAMP accumulation in EP2 S cells compared with wild-type and AS cells was similar to that observed in endometrial adenocarcinoma explants stimulated with PGE2, which we reported previously (9). We therefore believe that our Ishikawa EP2 receptor cell line is a good model system for investigating the molecular signal transduction pathways mediating the role of PGE2 in endometrial adenocarcinoma cells via the EP2 receptor, which may be causative toward the development and/or progression of endometrial tumors in vivo. Recently, the phenomenon of receptor tyrosine kinase transactivation by GPCRs has been described after the observation that suppression of EGFR kinase activity by the specific EGFR kinase inhibitor AG1478 strongly diminished the ERK1/2 MAPK activation that is mediated by GPCR agonists (31, 32, 33). New evidence has indicated that GPCR-mediated, and particularly prostanoid GPCR-mediated receptor tyrosine kinase transactivation may be crucial for transducing mitogenic signaling and tumor growth (24, 34, 35). This implies that multiple divergent intracellular signaling pathways are coactivated after prostanoid-receptor binding. We examined whether signaling via EP2 receptor in Ishikawa cells involved productive cross-communication with the EGFR signaling system, and found that PGE2 rapidly tyrosine phosphorylated the EGFR in Ishikawa EP2 S cells, compared with wild-type and EP2 AS cells. Several mechanisms are proposed for the transactivation of EGFR by GPCRs (32, 36, 37, 38). One of these mechanisms involves the activation of transmembrane MMP and extracellular release of heparin-binding EGF (HB-EGF) from its latent membrane-spanning precursor in the plasma membrane. Once cleaved, the HB-EGF ligand can associate with and activate the EGFR and induce ERK1/2 MAPK signaling. Alternatively, several reports have demonstrated that activation of the c-Src family of nonreceptor tyrosine kinases is involved in GPCR-mediated EGFR transactivation, by activation of intracellular protein-phosphorylation cascades (32, 36, 37, 38). To determine the mechanism of PGE2-EP2 receptor mediated transactivation of the EGFR, Ishikawa wild-type, EP2 S, and EP2 AS cells were cotreated with specific inactivators of PKA, c-Src, MMP, and EGFR kinase activity before stimulation with PGE2. PGE2-induced phosphorylation of EGFR was inhibited by inactivation of PKA, c-Src, and EGFR kinase, but not by treatment with a general MMP inhibitor. We must emphasize that caution must be exercised in the interpretation of data generated using inhibitors of signal transduction because these chemical compounds may be crossreactive with other signaling pathways. However, because significant inhibition of PGE2-induced phosphorylation of EGFR was observed with 10 µM of the PKA-specific inhibitor H-89, well below the Ki values for inhibition of protein kinase C, myosin light chain kinase, Ca2+/calmodulin kinase II, and casein kinase I and II (Ki > 25 µM) (39); 10 µM PP2, a potent inhibitor of the c-Src family of nonreceptor tyrosine kinases used well below the IC50 values for inhibition of JAK2/ZAP-70 (IC50 > 50 µM) (40) and in our study had no significant inhibition of EGF-induced phosphorylation of EGFR; and 100 nM AG1478, a selective inhibitor of EGFR kinase used well below the IC50 for inhibition of c-erbß2 or erythroblastosis virus (also called HER2-neu) (IC50 > 100 µM) and platelet-derived growth factor receptor kinase (IC50 > 100 µM) (41), this suggests to us that the observed inhibition of EGFR phosphorylation with these specific inhibitors, after treatment of Ishikawa cells with PGE2, was not due to inhibition of other signaling pathways. Moreover, treatment of EP2 S cells with dibutyryl cAMP (a cell-permeable cAMP analog) confirmed that EGFR phosphorylation in EP2 S cells occurs via cAMP-mediated activation of PKA, as EGFR phosphorylation in cells that were stimulated with dibutyryl cAMP was inhibited by cotreatment with inactivators of PKA and EGFR kinase. Treatment of EP2 S cells with EGF phosphorylated EGFR independently of PKA and c-Src. Cotreatment of EGF-stimulated cells with the PKA or Src inhibitor did not reduce the EGFR phosphorylation, confirming that PGE2-EP2 transactivation of the EGFR, unlike EGF-induced EGFR activation, occurs predominantly through activation of PKA and c-Src. Recently, Pai et al. (24) and Buchanan et al. (17) reported a similar finding in colon cancer cells, consistent with the idea that PGE2 transactivates the EGFR via an intracellular c-Src-mediated pathway.
The integrated response to activation of EGFR kinase results in numerous downstream signaling events being initiated. These include activation of small GTPases such as Rho, Rac, and Ras leading to activation of MAPKs and other downstream factors (42). GPCR-mediated activation of MAPK signaling is known to be a potent regulator of cell growth, differentiation and development (43). We examined whether activation of EGFR by PGE2 stimulates the downstream MAPK (ERK, p38, and JNK) pathways. We found that within our experimental paradigms, PGE2-induced activation of EGFR in Ishikawa cells was accompanied by a rapid increase in ERK activation (but not p38 or JNK). This PGE2-induced effect was significantly elevated in EP2 S cells compared with wild-type and EP2 AS cells. Cotreatment of cells with inhibitors of PKA, c-Src, and EGFR kinase (but not MMP) abolished the PGE2-induced phosphorylation of ERK1/2. Treatment of EP2 S cells with dibutyryl cAMP alone or in combination with inhibitors of PKA, c-Src, and EGFR kinase confirmed that cAMP-mediated ERK1/2 activation was mediated by PKA and subsequent c-Src and EGFR activation. In contrast, we found that the PKA or c-Src inhibitor, however, did not significantly inhibit EGF activation of ERK1/2 signaling. Recently, Fujino et al. (13) have demonstrated that PGE2 acts via the EP2 receptor in human embryonic kidney cells to activate T cell factor signaling via the cAMP-dependent PKA pathway (13). Interestingly, in the human embryonic kidney cell background, PGE2 interaction with the EP2 receptor was not associated with ERK activation (22). These data demonstrate that the function of PGE2 in EP2 receptor overexpressing cells may be cell-type-specific and would appear, at least in Ishikawa cells, that PGE2-mediated signaling via the EP2 receptor, occurs via a cAMP- and c-Src-mediated phosphorylation of the EGFR leading to subsequent activation of ERK1/2. From our data, it also appears that these effects of PGE2 upon the EGFR occur predominantly through an intracellular pathway. Our collective findings using chemical inhibitors and RNA interference are nevertheless consistent with a growing body of evidence, suggesting that transactivation of EGFR by GPCRs is a recurrent theme in cell signaling.
The up-regulation of angiogenic factors is one of the hallmarks of tumorigenesis. As the tumor’s demand for nutrients and oxygen increases, an increased vascularization is necessary to supply nutrients to the tumor. To sustain and facilitate growth, cancer cells produce a wide variety of factors, including basic fibroblast growth factor, VEGF, basic fibroblast growth factor-binding protein and platelet-derived growth factor at the site of tumor growth that create a proangiogenic environment and lead to angiogenesis, (7, 25). Our data using chemical inhibitors of signal transduction and RNA interference demonstrate the involvement of the EGFR and ERK1/2 signaling in the activation of VEGF expression and secretion in EP2 receptor overexpressing Ishikawa cells. Although it is possible that the MEK inhibitor could inhibit other convergent signaling pathways, the concentration that we have used in the present study is well below the IC50 values for inhibition of other serine/threonine kinases described (44). Interestingly, the cell-permeable cAMP analog, dibutyryl cAMP also up-regulated the expression of VEGF in EP2 S cells after 48 h of agonist treatment and these effects could be reversed by cotreatment of cells with the inhibitors of ERK1/2 and EGFR kinase (data not presented). These findings are similar to one of a number of recent studies where a cell permeable cAMP analog increased VEGF production in pancreatic cancer cells (18). Our combined data suggest that elevated EP2 receptor in endometrial adenocarcinoma cells promotes a proangiogenic environment predominantly via a cAMP-dependent transactivation of the EGFR and ERK1/2 pathways. VEGF is known to be the progenitor angiogenic factor in the formation of new blood vessels (45). In colon carcinoma cells, elevated synthesis of PGE2 results in the up-regulation of angiogenic factors, including VEGF, which exerts a paracrine function on endothelial cells to promote the arrangement of endothelial cells into tubular structures (7). It is therefore feasible to suggest that the VEGF, produced in and secreted from endometrial adenocarcinoma cells, would similarly exert a proangiogenic action upon adjacent endothelial cells to promote vascular branching and sprouting thus enhancing blood flow to the tumor, creating an environment to sustain tumor growth.
Taken together, these data suggest that targeted inhibition of EP2 and EGFR function in endometrial carcinomas could effectively block the signaling and transcription of target genes associated with angiogenesis. High EGFR levels in endometrial cancer have been correlated with poor histopathological grading, greater invasiveness and reduced patient survival (46, 47). Blockade of EGFR signaling with an orally active EGFR tyrosine kinase inhibitor has been used successfully in renal cell carcinomas in the kidney of nude mice to inhibit tumor angiogenesis, by reducing VEGF expression (48). Moreover, in a recent study, Torrance et al. (49) have demonstrated that the use of a nonselective COX enzyme inhibitor in combination with an inhibitor of EGFR kinase can reduce polyp formation in APC716 mice more effectively than either compound on their own (49). In light of these studies, a combination of EP2 receptor antagonist and EGFR kinase inhibitor may be an efficacious therapy for women with endometrial adenocarcinoma. Further studies are needed to evaluate the use of such combinatorial approaches targeted toward the prostanoid signaling and growth factor receptor signaling pathways as a means of therapy for cancer prevention or treatment. In conclusion, our data demonstrate that PGE2 promotes the expression and secretion of VEGF in endometrial adenocarcinoma cells overexpressing the EP2 receptor via the intracellular transactivation of the EGFR and activation of ERK1/2, through a PKA and c-Src-dependent mechanism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients and Tissue Collection
Endometrial adenocarcinoma tissue (poorly differentiated, n = 3; moderately differentiated, n = 3; and well differentiated, n = 4) was collected from women undergoing hysterectomy and who had been prediagnosed to have adenocarcinoma of the uterus. Hysterectomy specimens for adenocarcinoma were collected from theater and placed on ice. With minimal delay, the specimens were opened by a gynecological pathologist, and small samples (
5 mm–3 cm) of adenocarcinoma tissue were snap frozen in dry ice and stored at –70 C (for RNA extraction). The diagnosis of adenocarcinoma was confirmed histologically in all cases. All women with endometrial adenocarcinoma were postmenopausal. Ethical approval was obtained from Lothian Research Ethics Committee and written informed consent was obtained from all subjects before tissue collection.
Cell Culture
Human Ishikawa endometrial adenocarcinoma cells (European Collection of Cell Culture, Centre for Applied Microbiology, Wiltshire, UK) were maintained in DMEM nutrient mixture F-12 with glutamax-1 and pyridoxine, supplemented with 10% FCS, and 1% antibiotics (stock 500 IU/ml penicillin and 500 µg/ml streptomycin) at 37 C and 5% CO2 (vol/vol). Stable EP2 transfectant cells were maintained under the same conditions with the addition of a maintenance dose of 200 µg/ml G418.
EP2 Receptor Amplification and Cell Transfections
RNA was extracted from proliferative phase human endometrial tissue and reverse transcribed as described previously (50). The EP2 receptor was amplified from proliferative phase cDNA by PCR using standard PCR mix containing forward 5'-ATCTCTTTTCCAGGCACCCCAC-3' and reverse 5'-TTTTAAACTGACC-TCAAAGGTCAGC-3' primers. To amplify by PCR, sample mix was denatured at 94 C for 5 min and subjected to 40 cycles of 94 C for 1 min, 58 C for 1 min, and 68 C for 1 min, with a final extension step of 68 C for 7 min. After amplification, samples were cooled to 4 C and visualized on 1% agarose gels. The PCR product was ligated into the pCRII-TOPO vector (Invitrogen, De Schelp, The Netherlands) followed by sequencing in both directions using a PE Applied Biosystems (Warrington, UK) 373A automated sequencer. The EP2 receptor cDNA was ligated into the pcDNA3.1 (Invitrogen) expression vector in both S and AS directions, followed by sequencing (as already described) to confirm orientation. EP2 receptor cDNA was transfected into Ishikawa cells in the S and AS direction using superfect transfection reagent (QIAGEN, Crawley, UK) according to the manufacturer’s recommendations. Clones were selected for with addition of 800 µg/ml G418. Fifty single EP2 receptor S and AS clones were selected using cloning cylinders (Sigma) and screened by immunoblot analysis and cAMP ELISA. Three S (S32, S33, S41) and three AS (A4, A33, A39) clones, which demonstrated the greatest and least EP2 receptor expression and accumulation of intracellular cAMP in response to administration of 100 nM PGE2 respectively, compared with wild-type Ishikawa cells, were chosen for further experiments. All clones were characterized and exhibited identical phenotypic and biochemical alterations. The results of our studies using the S33 and A4 clones are presented here. Similar reproducible results were obtained using the other clones.
Immunofluorescent Microscopy
The site of EP2 receptor protein expression was localized in wild-type, S, and AS Ishikawa cells by immunofluorescence microscopy to determine whether all the EP2 receptor was trafficked to the plasma membrane compartment. Approximately 10,000 wild-type, S and AS cells were seeded in chamber slides, allowed to attach and grow overnight, before being fixed in 100% ice-cold methanol. After fixing, cells were washed in TBS [50 mM Tris-HCl, 150 mM NaCl (pH 7.4)] and blocked using 5% normal swine serum diluted in TBS. Subsequently, the cells were incubated with polyclonal rabbit anti-EP2 receptor antibody at a dilution of 1:50 at 4 C for 18 h. Control cells were incubated with rabbit IgG. Thereafter, the cells were incubated with secondary swine antirabbit tetramethyl-rhodamine-isothyocyanate (Dako Corp., High Wycombe, UK) at 25 C for 20 min. Cells were then mounted in permafluor (Immunotech-Coulter, Buckinghamshire, UK) and coverslipped. Fluorescent images were visualized and photographed using a Carl Zeiss (Jena, Germany) laser scanning microscope LM 510. The fluorophor was detected using the helium/neon 1 laser beam (excitation, 543 nm) and an emission band pass filter 560–615 nm.
Protein Extraction
For EGFR transactivation studies, 3 x 106 cells were seeded in 10-cm dishes and for MAPK studies 1 x 106 cells were seeded in 5-cm dishes. The following day, cells washed with PBS and incubated in serum-free culture medium containing penicillin/streptomycin (as described previously) and 8.4 µM indomethacin (a dual COX-enzyme inhibitor used to inhibit endogenous prostanoid biosynthesis) for at least 16 h. The next day, cells were pretreated with specific inhibitors for c-Src (PP2, 10 µM), MMP (GM6001, 10 µM), protein kinase A (PKA; H-89, 10 µM), EGFR kinase (AG1478, 100 nM), or MEK1/2 (PD98059, 50 µM) for 1 h before stimulation with 100 nM PGE2 (for the time period specified in the figure legends) or left as control. After stimulation with PGE2, cells were washed with ice-cold PBS. Proteins were extracted with a protein lysis buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris/HCl (pH 7.4), 1 mM EDTA, 5 mM EGTA, 0.1% sodium dodecyl sulfate containing 2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4 and 5 µg/ml aprotinin]. Thereafter, insoluble material was pelleted by centrifugation at 14,000 x g for 20 min at 4 C. The clarified lysate was removed to a new tube for protein quantification, SDS-PAGE, and Western blotting. The protein content in the supernatant fraction was determined using protein assay kits (Bio-Rad, Hemel Hempstead, UK).
Immunoprecipitation and Western Blot Analysis
For immunoprecipitation studies, equal amounts of protein were incubated with specific EGFR antibody preconjugated to protein A Sepharose overnight at 4 C with gentle rotation. Beads were washed extensively with lysis buffer and immune complexes solubilized in Laemmli buffer [125 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 20% glycerol, and 0.05% bromophenol blue] and then boiled for 5 min. For EP receptor expression in cells and MAPK studies, a total of 50 µg of protein was resuspended in 20 µl of Laemmli buffer. Proteins were resolved on 4–20% Tris-glycine gels (NOVEX, Invitrogen), transferred onto polyvinylidene difluoride membrane (Millipore, Watford, UK) and subjected to immunoblot analysis. Membranes were blocked for 1 h at 25 C in 4% BSA diluted in TBST (50 mM Tris-HCl, 150 mM NaCl and 0.05% vol/vol Tween 20) and incubated with specific primary antibodies. After washing and incubating with alkaline-phosphatase-conjugated secondary antibodies, immunoreactive proteins were visualized by the ECF chemiluminescence system according to the manufacturer’s instructions. Specific proteins were revealed and quantified by and normalized to total protein expression using STORM 860 PhosphorImager (Molecular Dynamics, Amersham Biosciences). All data are presented as mean ± SEM.
cAMP Assay
PGE2-induced cAMP accumulation was determined in response to administration of 100 nM PGE2 and was performed as described previously (25). Cells (2 x 105) were plated in six-well dishes and allowed to attach overnight. The following day, the cells were synchronized by incubating with fresh medium containing no fetal calf serum for 24 h. Thereafter, the culture medium was removed and replaced with serum-free medium containing 3-isobutyl-1-methyl xanthine (Sigma) to a final concentration of 1 mM for 60 min at 37 C. Cells were then stimulated with 100 nM PGE2 for 5, 10, or 15 min, respectively, or left unstimulated. After stimulation, cells lysed in 0.1 M HCl. cAMP concentration was quantified by ELISA using a cAMP kit (Biomol, Affiniti, Exeter, UK) according to the manufacturer’s protocol and normalized to protein concentration of the lysate. Protein concentrations were determined using protein assay kits (Bio-Rad). Data are presented as mean ± SEM.
Taqman Quantitative RT-PCR
VEGF and EP2 receptor expression in Ishikawa cells and EP2 expression in endometrial adenocarcinomas was measured by quantitative RT-PCR analysis. For VEGF and EP2 receptor expression in Ishikawa cells, 5 x 105 cells were seeded in 5-cm dishes and allowed to attach and grow overnight. The following day, cells were synchronized by serum withdrawal for 12 h in serum-free medium containing 8.4 µM indomethacin. Thereafter, medium was removed and replaced with fresh medium containing indomethacin with either 100 nM PGE2, vehicle, 5 mM dibutyryl cAMP or PGE2/dibutyryl cAMP and AG1478 or PGE2/dibutyryl cAMP and PD98059 for 48 h. After 24 h, fresh PGE2/dibutyryl cAMP or vehicle and inhibitor was added to the culture medium. RNA was extracted from cells using Tri-reagent (Sigma) after the manufacturer’s guidelines. Endometrial adenocarcinoma tissues (n = 4, poorly differentiated; n = 4, moderately differentiated; n = 2, well differentiated) were processed as described earlier (9). Once extracted and quantified, RNA samples were reverse transcribed using MgCl2 (5.5 mM), deoxynucleotide triphosphates (0.5 mM each), random hexamers (2.5 µM), ribonuclease inhibitor (0.4 U/µl), and multiscribe reverse transcriptase (1.25 U/µl; all from PE Biosystems, Warrington, UK). The mix was aliquoted into individual tubes (16 µl/tube) and template RNA was added (4 µl/tube of 100 ng/µl RNA). After mixing, samples were incubated for 90 min at 25 C, 45 min at 48 C and 95 C for 5 min. Thereafter, cDNA samples were stored at –20 C. A tube with no reverse transcriptase was included to control for DNA contamination.
To measure cDNA expression a reaction mix was prepared containing Taqman buffer [5.5 mM MgCl2, 200 µM deoxy (d) ATP, 200 µM dCTP, 200 µM dGTP, 400 µM deoxyuridine triphosphate], ribosomal 18S forward and reverse primers and probe (50 nM), forward and reverse primers for VEGF/EP2 (300 nM), VEGF/EP2 probe (100 nM), AmpErase UNG (0.01 U/µl) and AmpliTaq Gold DNA Polymerase (0.025 U/µl; PE Biosystems). After mixing, 48 µl were aliquoted into separate tubes and 2 µl/replicate (40 ng) of cDNA added and mixed before placing duplicate 24-µl samples into a PCR plate. A no-template control (containing water) was included in triplicate. PCR was carried out using an ABI Prism 7700. VEGF and EP2 primers and probe for quantitative PCR were designed using the PRIMER express program (PE Biosystems). The sequence of the VEGF primers and probe were as follows: forward, 5'-TAGCTGCGCTGATAGACAT-3'; reverse, 5'-TACCTCCACCATGCCAAGT-3'; probe (FAM labeled, 6-carboxyfluorescein) 5'-ACTTCGTGATGATTCTGCC-3'. The sequence of the EP2 receptor primers and probe were as follows: EP2 forward, 5'-GAC CGC TTA CCT GCA GCT GTA C-3'; EP2 reverse, 5'-TGA AGT TGC AGG CGA GCA-3'; EP2 probe (FAM labeled, 6-carboxyfluorescein): 5'-CCA CCC TGC TGC TGC TTC TCA TTG TCT-3'. The ribosomal 18S primers and probe sequences were as follows: forward, 5'-CGG CTA CCA CAT CCA AGG AA-3'; reverse, 5'-GCT GGA ATT ACC GCG GCT-3'; probe, (VIC-labeled, PE Biosystems) 5'-TGC TGG CAC CAG ACT TGC CCT C-3'. Data were analyzed and processed using Sequence Detector version 1.6.3 (PE Biosystems). Expression of VEGF/EP2 was normalized to RNA loading for each sample using the 18S ribosomal RNA as an internal standard. Results are expressed as relative expression to an internal standard RNA.
RNA Interference
siRNA duplexes were used to abolish EGFR expression and function using an EGFR siRNA/siAB assay kit (Upstate, Woverton Mill South, Milton Keynes, UK). Ishikawa S cells were seeded to a density of 1 x 105 cells per well in six-well dishes and exposed to 100 nM EGFR siRNA or control random siRNA in the presence of superfect (QIAGEN) for 4 h and then cultured for 24 h in complete medium. Thereafter, cells were serum-starved for 12 h in medium containing indomethacin and then exposed to either 100 nM PGE2 or vehicle for 48 h. Thereafter, cells were lysed with either Tri-Reagent and subjected to quantitative RT-PCR analysis or protein lysis buffer and subjected to Western blot analysis as described earlier. Culture medium was removed from cells before lysis for secreted VEGF analysis. Cells were maintained with 8.4 µM indomethacin throughout to abolish any endogenous production of prostanoids.
VEGF ELISA
Secreted VEGF was measured by ELISA. Cells were first synchronized by serum withdrawal for 12 h in serum-free medium containing 8.4 µM indomethacin. Thereafter, medium was removed and replaced with fresh complete medium contain 100 nM PGE2 or vehicle and indomethacin for 48 h. Culture medium was removed and VEGF protein was measured using a Human VEGF ELISA kit as per the manufacturer’s instruction (Oncogene, Beeston, Nottingham, UK). Cells were lysed and protein concentration determined using protein assay kits (Bio-Rad) and total VEGF secreted was normalized per mg of protein in the lysate. Data are expressed as percentage above basal where the amount of VEGF secreted in treated cells is divided by the amount secreted in cells treated with the vehicle or vehicle plus inhibitor. The data are presented as mean ± SEM from four independent experiments.
Statistics
Where appropriate, data were subjected to statistical analysis with ANOVA and Fisher’s protected least significant difference tests (Statview 5.0; Abacus Concepts Inc., Carpinteria, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: COX, Cyclooxygenase; DMSO, dimethylsulfoxide; EGFR, epidermal growth factor receptor; EP1–4, E-series prostanoid receptors; GPCR, G protein-coupled receptor; HB-EGF, heparin-binding EGF; JNK, c-Jun N-terminal kinase; MMP, matrix metalloproteinase inhibitor; PG, prostaglandin; PKA, protein kinase A; siRNA, small inhibitory RNA molecules; VEGF, vascular endothelial growth factor.
Received for publication January 19, 2004. Accepted for publication March 18, 2004.
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