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Published as doi: 10.1096/fj.06-7581com.
 (The FASEB Journal. 2007;21:2418-2430.)
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EP2 prostanoid receptor promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways

Sandra Donnini*,1, Federica Finetti*,1, Raffaella Solito*, Erika Terzuoli*, Andrea Sacchetti{dagger}, Lucia Morbidelli*, Paola Patrignani{dagger} and Marina Ziche*,2

* Department of Molecular Biology, Pharmacology Angiogenesis Laboratory, University of Siena, Siena, Italy; and

{dagger} Department of Pharmacology, University of Chieti, Chieti, Italy

2Correspondence: Department of Molecular Biology, Pharmacology Angiogenesis Lab., University of Siena, Via Aldo Moro, 2, 53100, Siena, Italy. E-mail: ziche{at}unisi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In squamous cell carcinoma, the levels of nitric oxide (NO) derived from inducible NO synthase (iNOS) and prostaglandin E2 (PGE2) derived from cyclooxygenase-2 (COX-2) originated from tumor cells or tumor-associated inflammatory cells have been reported to correlate with tumor growth, metastasis, and angiogenesis. The present study examined the role of the iNOS signaling pathway in PGE2-mediated tumor invasiveness and proliferation in squamous cell carcinoma, A431, and SCC-9 cells. Cell invasion and proliferation promoted by PGE2 were blocked by iNOS silencing RNA or iNOS/guanylate cyclase (GC) pharmacological inhibition. Consistently, iNOS-GC pathway inhibitors blocked mitogen-activated protein kinase-ERK1/2 phosphorylation, which was required to mediate PGE2 functions. In vivo, in A431 cells implanted in nude mice, GC inhibition also decreased the tumor proliferation index and ERK1/2 activation. PGE2 effects were confined to the selective stimulation of the EP2 receptor subtype, leading to epidermal growth factor receptor (EGFR) transactivation via protein kinase A (PKA) and c-Src activation. EP2-mediated ERK1/2 activation and cell functions were abolished by inhibitors of PKA, c-Src, and EGFR, as well as by inhibiting iNOS pathway. Silencing of iNOS also impaired EGFR-induced ERK1/2 phosphorylation. These results indicate that iNOS/GC signaling is a downstream player in the control of EP2/EGFR-mediated tumor cell proliferation and invasion.—Donnini, S., Finetti, F., Solito, R., Terzuoli, E., Sacchetti, A., Morbidelli, L., Patrignani, P., Ziche, M. EP2 prostanoid receptor promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways.


Key Words: Key Words: nitric oxide • prostaglandin E2 • invasiveness


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PROGRESSION OF SQUAMOUS CELL CARCINOMA such as head and neck carcinoma correlates with the spread of distant metastasis and loco-regional recurrences (1) . In turn, angiogenesis (i.e., the increased formation of new vessels) is perhaps the most important determinant of tumor growth and metastasis spread (2) . In a previous report investigating head and neck cancer patients, we have shown that microvessel density of the tumor periphery, and particularly the invasive edge of the tumor, strongly correlated with the increased expression of inducible nitric oxide synthase (iNOS) protein and iNOS activity (3) . The notion that iNOS is a key player in tumor development, widely documented for a variety of solid tumors, is now a mainstay of cancer biology (3 4 5 6) . Recent reports have emphasized the importance of inducible cyclooxygenase (COX-2) and its products for the progression of a variety of tumors including prostate and colon, head and neck, ovarian tumors, and hepatocellular carcinoma (7 8 9 10 11 12) . Overexpression of COX-2 has been documented to be associated with invasive metastatic process, resulting in a poor prognosis for cancer patients (13) . There is also clear evidence for a close interplay between iNOS and COX-2 proteins that extends to their respective products, nitric oxide (NO) and the prostanoid prostaglandin E2, PGE2 (14 15 16) . Thus, the COX-2 overexpression observed in tumors may be viewed as part of the concerted process leading to increased angiogenesis and metastatic spread (17) . Most tumors that intensely express COX-2 enzyme have also been found to contain high levels of PGE2 (18 , 19) .

PGE2 exerts its autocrine/paracrine effects on target cells by coupling to four subtypes of G-protein-coupled receptors classified as EP1, EP2, EP3, and EP4 (E-series prostanoid receptors) (20 , 21) . These receptors are often coexpressed in the same cell type and use different, and in some cases, opposing intracellular signaling pathways (22 23 24 25 26) . Studies reported the crucial role played in particular by EP2 or EP4 receptor signaling in mediating tumor progression and/or tumor-associated angiogenesis (27 28 29) . Recently, it was shown that PGE2 stimulation of both EP2 and EP4 receptors involves transactivation of the epidermal growth factor receptor (EGFR) signaling pathway to promote tumorigenesis (30 31 32) . EGFR phosphorylation leads to activation of downstream signaling molecules involved in cell proliferation and survival pathways of the ras/raf/MAPK and PI3K/Akt, respectively (33 34 35) .

In squamous cell carcinoma, overexpression of EGFR has been correlated with increased levels of COX-2 and iNOS activity, PGE2 and NO production, angiogenesis, and metastasis; this in turn has been correlated with a poor prognosis and resistance to therapy (2 , 36 , 37) . To date, a direct cross-linking between iNOS, COX-2/PGE2, and EGFR has not been elucidated.

In the present study the squamous carcinoma cells, A431 and SCC-9, were used to delineate the role of iNOS/guanylate cyclase (GC) signaling pathway on prostanoid-mediated stimulation of tumor cell invasion and growth. Moreover, in PGE2-mediated A431 cell invasion and growth, the role of MAPK-ERK1/2 was investigated, and the potential link between iNOS/GC signaling and ERK1/2 activity was elucidated.

Results indicate that PGE2-induced promotion of A431 tumor invasion and growth requires activated iNOS-CG and MAPK-ERK1/2, and is mediated by EGFR transactivation via EP2/PKA and the c-Src signaling pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
Human epidermoid carcinoma A431 and SCC-9 cells were obtained by ATCC (Manassas, VA, USA). Cells were maintained in culture in DMEM (A431) or in F12:DMEM (SCC-9) supplemented with 4500 mg/L glucose, 2 mM L-glutamine, antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) and 10% fetal calf serum (FCS) (Hyclone, Logan, UT, USA). All media and antibiotics for cell culture were purchased from Sigma (St. Louis, MO, USA). Cells were split 1:8 (A431) or 1:5 (SCC-9) twice a week.

Cell proliferation assay
Cell proliferation was quantified by Vybrant MTT cell proliferation assay kit (Molecular Probes, Carlsbad, CA, USA). A431 cells (2.5x103) or SCC-9 (3x103) were seeded in 96-multiwell plates in medium with 10% serum for 18 h, starved in 0.1% FCS for 24 h, then exposed to PGE2 (Sigma), NOC-12 (Calbiochem-Novabiochem, La Jolla, CA, USA), or PGE2 receptor subtype agonists (17-phenyl trinor prostaglandin E-2, Butaprost, sulprostone, and prostaglandin E-1 alcohol, obtained from Cayman Chemicals, Ann Arbor, MI, USA) for 48 h in 0.1% FCS. Where indicated, cells were pretreated with NOS pathway inhibitors L-nitro-M-methyl arginine, L-NMMA (Sigma), and 1H-(1, 2, 4)oxadiazolo[4,3-a]quinoxalin-1-one, ODQ (Alexis Biochemicals, Lausen, Switzerland) or the PKA inhibitor H89 (Calbiochem-Novabiochem), the MEK inhibitor U0126 (Calbiochem-Novabiochem), or the EGFR inhibitor AG1478 (Calbiochem-Novabiochem) for 40 min in 0.1% FCS, then stimulated with PGE2, NOC-12, or the EP agonists. After 44 h, medium was removed and cells were incubated for 4 h with fresh medium in the presence of 1.2 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Living cells reduce MTT to a strongly pigmented formazan product. After solubilization in DMSO, absorbance of the formazan was measured with a microplate absorbance reader (Tecan, San Jose, CA, USA) at 540 nm. Data are reported as 540 nm absorbance/well.

Invasion assay
Cell invasion was investigated using the modified Boyden chamber as described previously with minor modifications (38) . Briefly, 3 x 105 cells were seeded in a 6 cm dish in 10% FCS for 18 h, starved in 0.1% for 24 h, then trypsinized and stimulated for 40 min with or without the NOS pathway inhibitors or PKA, MEK, or EGFR inhibitors in 0.1% FCS. Cell suspension (1.2x104 cells) was added to the upper wells. PGE2 or NOC-12 was used as chemoattractive molecules. Invasion was measured in triplicate by counting the number of cells that had moved in 16 h across the filter coated with Matrigel (250 µg/ml). Cells were counted in five random fields/well at a magnification of 400x. Data are reported as total number of cells counted/well.

Western blot analysis
The procedure was performed as described previously (39 , 40) . Electrophoresis was carried out in SDS/10% polyacrylamide gels for iNOS, ERK1/2, and EP receptors and in SDS/8% polyacrylamide gels for phospho-tyrosine, EGFR, or anti-PKA C-{alpha}. Membranes were incubated for 18 h at 4°C with mouse monoclonal primary antibody directed against iNOS (Cayman Chemicals, Ann Arbor, MI, USA), human phospho-ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-EP receptors (anti-EP1, EP2, and EP3 Cayman Chemicals, USA; anti-EP4 was kindly provided by Drs. M. D. Breyer and R. M. Breyer, Vanderbilt University Medical School, Nashville, TN, USA), antiphospho-tyrosine (P-Tyr-100) (Cell Signaling Technology), or anti-EGFR or anti-PKA C-{alpha} (Cell Signaling Technology, Inc.), diluted 1:2000 (anti-P-ERK1/2 and anti-PKA C-{alpha}) and 1:1000 (anti-EP receptors, anti-P-Tyr, anti-EGFR, and anti-iNOS) in PBS. The primary antibody was detected by incubating the membranes for 1 h with horseradish peroxidase-HRP conjugated rabbit anti-mouse secondary antibody (Promega, Madison, WI, USA) or goat anti-rabbit (Sigma) diluted 1:2000, and 1:2500, respectively, in PBS, followed by enhanced chemiluminescence system for detection (Amersham, Arlington Heights, IL, USA). Images were digitalized with CHEMI DOC Quantity One program, blots were analyzed in duplicate by densitometry using NIH Image 1.60B5 software, and the optical density (OD) values were normalized to OD for total EGFR (phospho-tyrosine blots), total ERK1/2 (phospho-ERK1/2 blots), or actin (EP receptors and iNOS).

siRNA transfection
21-Nucleotide RNAs were chemically synthesized by Qiagen (Milan, Italy). Synthetic oligonucleotides were deprotected and gel-purified as described (41) . The siRNA sequence targeting human iNOS (5'-AACCCAGCTGCTGCTCCAAAA-3') corresponded to region 2661–2681 relative to the start codon, and targeting human PKACA (5'-AAGCCGGAGAATCTGCTCATT-3') corresponded to region 704–724 relative to the start codon. Control siRNA is a random siRNA provided by Qiagen (Valencia, CA, USA). For annealing of siRNAs, 20 µM single strands were incubated in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, and 2 mM magnesium acetate) for 1 min at 90°C followed by 1 h at 37°C. The day before transfection, cells were trypsinized and 3 x 105 cells were seeded in 6-well plates. Transient transfection of siRNA was carried out using lipofectamine (Invitrogen, Carlsbad, CA, USA). OPTIMEM medium (100 µl) (Life Technologies, Inc., Gaithersburg, MD, USA) and 10 µl lipofectamine per well were preincubated for 5 min at room temperature. During the period for this incubation, 100 µl OPTIMEM medium was mixed with 10 µg siRNA. The two mixtures were combined and incubated for 20 min at room temperature for complex formation. After incubation, the entire mixture was added to the cells in a final concentration of 300 nM siRNAs. Cells were assayed 48 h after transfection.

Immunohistochemistry
After transfection with siRNA for iNOS, the cells were washed in PBS, fixed in acetone at –20°C, and washed again extensively. After blocking unspecific binding sites in 3% bovine serum albumin (BSA), the cells were incubated overnight at 4°C with the monoclonal antibody anti-iNOS (BD Transduction Laboratories, San Jose, CA, USA) diluted 1:30 in PBS containing 0.5% BSA. After the primary antibody, the cells were washed and incubated for 2 h with an isothiocyanate-labeled goat anti-mouse IgG secondary antibody (Sigma) diluted 1:50 in PBS with 0.5% BSA and mounted in Moviol 4–88 (Calbiochem).

NOS activity
NOS activity was measured as described previously (42) . Briefly, 5 x 105 A431 cells in 60 mm culture dishes were stimulated for 4 h with 50 µg/ml LPS, 1 µM PGE2, or 10 ng/ml EGF and treated as described (3) . All determinations were performed in duplicate. NOS activity is expressed as picomoles of [3H]citrulline formed/min per mg protein (pmol/min per mg proteins).

In vivo studies
Experiments have been performed in accordance with the guidelines of the European Economic Community for animal care and welfare (EEC Law No. 86/609) and the National Ethical Committee. To assess the in vivo effect of NOS pathway inhibition on A431 tumor growth, female immunodeficient mice (5- to 8-wk-old CD-1 nude mice, Charles River, Wilmington, MA, USA) were s.c. inoculated in the right flank with 107 A431 cells in a volume of 50 µl. After 10 days, when tumors reached a volume of 300 mm3, animals were randomly assigned to two different experimental protocols (5 mice per group). At this time peri-tumor treatment (i.e., injection of the ODQ, 10 µM/day/mice) or vehicle (5% DMSO in PBS) close to the tumor mass began. Daily treatment was performed for 4 consecutive days. Animals were observed daily for signs of cytotoxicity and sacrificed by CO2 asphyxiation. At day 5 animals were sacrificed and each tumor was split into two parts: one part was immediately frozen in liquid nitrogen for measurement of cGMP content; the other part was embedded in Tissue-Tek O.C.T. (Sakura, San Marcos, CA, USA), cooled in isopentane, and frozen in liquid nitrogen for histology. cGMP levels were measured in tissue homogenates by radioimmunoassay as described (3) . Seven µm-thick cryostat sections from tissue samples were stained with hematoxylin and eosin, and adjacent sections were used for immunohistochemical staining with the anti-COX-2 (Cayman), anti-microsomial prostaglandin E synthase-1 (m-PGES-1) (Cayman), anti-EP2 receptor (Cayman), anti-Ki67 (Chemicon, Temecula, CA, USA), and antiphospho-ERK1/2 (Cell Signaling) antibodies. Cryostat sections were first fixed in acetone –20°C and incubated for 10 min in 3% H2O2, washed (3x5 min) in TBS, then incubated in a blocking reagent (KIT Immunoperoxidase Secondary Detection System, Chemicon). Primary antibodies were then applied as follows: mouse monoclonal anti-Ki67 diluted 1:100 in PBS, 0.05% BSA; mouse monoclonal anti-COX-2, diluted1:100 (5 µg/ml) in PBS, 0.05% BSA; rabbit polyclonal anti-m-PGES-1 diluted 1:100 (5 µg/ml) in TBS, 0.05% BSA; mouse monoclonal anti-EP2 diluted 1:100 in TBS, 0.05% BSA, mouse monoclonal anti-pERK diluted 1:100 in TBS, 0.05% BSA. All the antibodies were applied for 1 h at room temperature. Sections were then washed (3x5 min in TBS) and incubated for 10 min in the appropriate species-specific biotinylated secondary antibodies (goat anti-mouse and goat anti-rabbit IgG, KIT Immunoperoxidase Secondary Detection System, Chemicon). After washing (3x5 min in TBS), the sections were incubated for 10 min in streptavidin-conjugated HRP. After this incubation, sections were exposed to 3,3-diaminobenzidine tetrahydrocloride for 8 min to produce a brown reaction product. Sections were then counterstained in hematoxylin and mounted in Aquatex (Merck, Rahway, NJ, USA). Western blot analysis for phosphotyrosine and EGFR was performed as reported above (see Western blot analysis).

Statistical analysis
Results are expressed as means ± SE. Statistical analysis was performed using Student’s t test and analysis of variance (ANOVA). When a significant difference was detected, multiple comparison analysis was performed using the Student-Newman-Keuls test. A value of P < 0.05 was considered to denote statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PGE2 promotes invasion and proliferation of squamous cell carcinoma via ERK1/2 activation
Squamous cell carcinoma responds to exogenous stimuli by increasing the synthesis of PGE2 (36) . We wanted to study whether this mediator would change functional parameters such as invasiveness and growth in A431 and SCC-9 cells, models of squamous cell carcinoma. PGE2 promoted cell invasiveness and growth in a concentration-related manner, maximal stimulation being obtained at 100 nM (Table 1 ). Further, we investigated the role of ERK1/2, a signal typically involved in cell growth, in PGE2-induced invasiveness and proliferation of squamous cell carcinoma. PGE2 (l µM, 10 min stimulation) strongly enhanced ERK1/2 phosphorylation, a signal that was reduced by U0126 (10 µM), the specific MEK inhibitor (Fig. 1 A, B). U0126 also suppressed invasion and proliferation of A431 and SCC-9 promoted by PGE2 (Fig. 1C-F ), demonstrating that ERK1/2 activation mediates PGE2 cellular functions.


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  		Table 1. Effect of exogenous PGE2 on A431 and SCC-9 tumor cell invasion and growtha


Figure 1
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  		Figure 1. ERK1/2 activity promoted by PGE2 mediates cell growth and invasion. A, B) Western blot analysis of ERK1/2 in response to PGE2 (1 µM, 10 min) in the presence or absence of the MEK inhibitor U0126 (10 µM, 40 min). For Western blot analysis, a representative gel of three with similar results is reported. Ratio = optical density of phospho-ERK1/2/total ERK1/2. Effect of U0126 (10 µM, 40 min) on A431 (C, E) and SCC-9 (D, F) cell proliferation and invasion induced by PGE2 (1 µM). Data reported as total cells counted/well for invasion and as absorbance (540 nm)/well for MTT assay are the means ± SE of 3 experiments run in triplicate. ***P < 0.001 vs. control response, ###P < 0.01vs. PGE2 stimulation.

PGE2 transactivation of EGFR mediates squamous cell carcinoma growth and invasion
Since PGE2 effects on tumor progression have been demonstrated to be mediated by transactivation of the EGFR (30 31 32) , we investigated whether PGE2 was able to promote EGFR transactivation in squamous cell carcinoma and whether it was the obligatory step in mediating the PGE2 responses. We assessed EGFR phosphorylation and cellular functions promoted by PGE2 in the presence or absence of AG1478, the selective EGFR tyrosine kinase inhibitor. EGFR tyrosine phosphorylation was significantly increased by PGE2 (l µM, 5 min stimulation, Fig. 2 A). Consistently, EGFR phosphorylation was sensitive to AG1478, leading to suppression of PGE2-mediated cell growth (Fig. 2A, B ). Similar results were obtained for invasion (data not shown).


Figure 2
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  		Figure 2. PGE2 transactivates EGFR. A) Representative Western blot showing EGFR phosphorylation induced by PGE2 (1 µM, 5 min) in the presence or absence of the selective EGFR inhibitor AG1478 (10 µM) in A431. Cells were pretreated with AG1478 for 40 min before application of PGE2. The gel is representative of three gels with similar results. Ratio = optical density of phospho-tyrosine/total EGFR. B) Cell proliferation induced by PGE2 (1 µM) in the presence or absence of AG1478 (1 µM, 40 min). SCC9 cells exposed to the same conditions gave similar results. Data reported as absorbance (540 nm)/well for MTT assay are the means ± SE of 4 experiments run in triplicate. ***P < 0.001 vs. control response, ###P < 0.01 vs. PGE2 stimulation.

EP2/PKA/c-Src pathway mediates EGFR transactivation and the cellular effects of PGE2
Next, we investigated whether the transactivation of EGFR by PGE2 could be assigned to a specific EP receptor subtype. First, we verified the presence of receptor subtypes in the squamous cell carcinoma model. Western blotting revealed the constitutive presence of EP1, EP2, EP3, and EP4 receptors in A431 cells (Fig. 3 A) and in SCC-9 cells (data not shown). EGFR tyrosine phosphorylation was assessed after exposure of cells to each subtype selective agonist (17-phenyl trinor prostaglandin for EP1/EP3, Butaprost for EP2, sulprostone for EP3, prostaglandin E-1 alcohol for EP4), all at 1 µM for 5 min. Since only EP2 stimulation with Butaprost increased EGFR phosphorylation to an extent similar to that attained by PGE2, we conclude that, at least for A431 cells, the transactivation of EGFR may be confidently assigned to the EP2 receptor subtype (Fig. 3B ). Consistently, AG1478 suppressed transactivation of EGFR (Fig. 3C ).


Figure 3
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  		Figure 3. PGE2 transactivates EGFR through EP2 receptor stimulation and PKA activation. A) Representative Western blot showing expression of the four receptor subtypes in A431 tumor cell line, EP1, EP2, EP3, and EP4. B) Western blot analysis of the EGFR phosphorylation induced by the agonists of the EP receptor subtypes (EP1, 17-phenyl trinor prostaglandin E-2, EP2, Butaprost; EP3, sulprostone; EP4, prostaglandin E-1 alcohol, all used at 1 µM) after 5 min stimulation. The gel is representative of two with similar results. Ratio = optical density of phosphotyrosine/total EGFR. Western blot analysis of the EGFR activation mediated by 1 µM Butaprost (C) or PGE2 (D) in the presence or absence of PKA inhibitor H89 (500 nM) or the selective EGFR inhibitor AG1478 (1 µM). A431 cells were pretreated 40 min with the inhibitors before stimulation for 5 min with Butaprost or PGE2. The gels are representative of three with similar results. Ratio = optical density of phospho-tyrosine/total EGFR.

Since PKA has been reported to mediate PGE2 intracellular signaling after activation of EP2 receptor subtype (21) , we investigated its role in PGE2/EP2-mediated EGFR transactivation as well as in PGE2 cell functions. Incubation with H89 (500 nM), a PKA blocker (43 , 44) , abolished both PGE2 and Butaprost-promoted EGFR phosphorylation (Fig. 3C, D ), reduced PGE2-induced ERK1/2 phosphorylation, and affected functional parameters in an A431 tumor cell line (Fig. 4 A, B). Similar results were obtained with SCC-9 (data not shown). To confirm the requirement of PKA activation for EGFR phosphorylation, we also silenced PKA activity by siRNA technology. Reduction of PKA expression in A431 significantly inhibited PGE2-induced EGFR phosphorylation, leaving EGF activity unchanged (Fig. 5 A). Similar results were obtained for SCC-9. These results indicate that EGFR transactivation is required for the full expression of PGE2 actions in these cells.


Figure 4
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  		Figure 4. PGE2-induced cell growth and invasion are mediated by PKA activation. A) Western blot analysis of ERK1/2 in response to PGE2 (1 µM, 10 min) in the presence or absence of the PKA inhibitor H89 (500 nM, 40 min). For Western blot analysis, a representative gel of three with similar results is reported. Ratio = optical density of phospho-ERK1/2/total ERK1/2. Effect of H89 (500 nM, 40 min) on A431 (B, C) cell proliferation and invasion induced by PGE2 (1 µM). Data, reported as total cells counted/well for invasion and as absorbance (540 nm)/well for MTT assay, are the means ± SE of 3 experiments run in triplicate. ***P < 0.001 vs. control response, ###P < 0.01 vs. PGE2 stimulation.


Figure 5
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  		Figure 5. PKA and c-Src are essential for PGE2-induced EGFR transactivation. A) Representative Western blot showing EGFR phosphorylation induced by PGE2 (1 µM, 5 min) or EGF (10 ng/ml, 5 min) in A431 cells silenced for 48 h for PKA. Si-PKA, cells treated with si-RNA for PKA; si-Control, cell treated with a random siRNA. B) Representative Western blot showing the EGFR phosphorylation induced by PGE2 (1 µM, 5 min) in the presence or absence of the c-Src inhibitor PP1 (500 nM). A431 cells were pretreated with PP1 for 40 min before PGE2 application. The gels are representative of three gels with similar results. Ratio = optical density of phospho-tyrosine/total EGFR.

In addition, EGFR pathway stimulation appeared to be dependent on c-Src activity, as application of its inhibitor (PP1, 500 nM) abolished PGE2-promoted phosphorylation of EGFR (Fig. 5B ).

The results clearly suggest that PGE2-mediated transactivation of EGFR is both PKA and c-Src dependent.

iNOS/GC signaling mediates PGE2 cellular and molecular effects in squamous cell carcinoma
As squamous cell carcinoma growth and invasion promoted by PGE2 appear to be regulated through EGFR transactivation and because in these tumors overexpression of EGFR and COX-2 has been correlated with increased levels of iNOS activity, we examined whether there was interplay between these signals. First, we analyzed the modulation of iNOS signaling pathway by measuring functional effects of PGE2 (1 µM) in A431 cells after iNOS silencing by siRNA technology (Fig. 6 A, B). Silencing of iNOS inhibited both basal cGMP production by 40% (data not shown), and suppressed PGE2-induced invasion and proliferation (Fig. 6C, D ). Transfection with the siRNA control did not change PGE2-induced functions (Fig. 6C, D ). Pharmacological inhibitors of the iNOS-GC pathway (L-NMMA, 200 µM, or the GC inhibitor ODQ,10 µM) also inhibited PGE2-induced growth and invasion, as well as ERK1/2 activity, leaving control responses intact (Fig. 6E-G ). These results emphasize the requirement of a functional iNOS/GC signaling for the expression of PGE2 effects in squamous cell carcinoma.


Figure 6
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  		Figure 6. Effect of iNOS pathway inhibition on squamous cell carcinoma invasion and growth. A) iNOS expression after 48 h mRNA-iNOS silencing in A431 cells was evaluated by Western blot analysis and B) immunohistochemistry. For Western blot, a representative gel of three with similar results is reported. Immunofluorescencent staining for iNOS was performed in A431 tumor cells treated with si-iNOS mRNA (central panel) or si-Control mRNA (right panel) or in untreated cells (left panel). Representative pictures demonstrate complete inhibition in iNOS expression (central panel) relative to those in the controls (left and right panels). C) Effect of si-iNOS RNA on PGE2 (1 µM) -induced A431 cell proliferation and D) invasion. Effect of the NOS inhibitor L-NMMA (200 µM, 40 min) and the GC inhibitor ODQ (10 µM, 40 min) on 1 µM PGE2-induced proliferation (E), invasion (F), and ERK1/2 activity (G) in A431. The effect of NOC-12 (1 µM), a NO donor drug, is shown as a positive control. Data are the means ± SE of 3 experiments run in triplicate. Data are reported as total cells counted/well for invasion or as absorbance (540 nm)/well for MTT assay. ***P < 0.001 vs. control response (0.1% serum), ##P < 0.01 and ###P < 0.001 vs. PGE2 stimulation.

The involvement of iNOS/GC signaling in transducing EP2/PGE2 effects was also documented by studying the effect of Butaprost (1 µM), which promoted growth, invasion, and ERK1/2 activation in A431 tumor cells (Fig. 7 A–C). These responses were suppressed by pretreatment with AG1478 (1 µM), U0126 (10 µM), or NOS/GC signaling pathway inhibitors (Fig. 7A, B ) as well as by si-iNOS mRNA (Fig. 7C ). These data underscore the relevance of iNOS/GC signaling in EP2/PGE2-mediated functions via EGFR transactivation.


Figure 7
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  		Figure 7. EP2 receptor mediates PGE2 effects on A431 cells. A) A431 cell proliferation and B) invasion in response to EP2 receptor agonist (1 µM Butaprost) in the presence or absence of L-NMMA (200 µM), ODQ (10 µM), U0126 (10 µM), or AG1478 (1 µM). Cells are pretreated for 40 min with the inhibitors before agonist stimulation. Data are reported as total cells counted/well for invasion or as absorbance (540 nm)/well for MTT assay; they are the means ± SE of 3 experiments run in triplicate. ***P < 0.001 vs. control response, #P < 0.5, ###P < 0.001 vs. Butaprost alone. C) Effect of si-iNOS RNA (48 h silencing) on ERK1/2 activity induced by Butaprost (1 µM, 10 min stimulation). A representative gel of two with similar results is reported. ##P < 0.01 Ratio = optical density of phospho-ERK1/2/total ERK1/2.

iNOS activity regulates PGE2/EGF/EGFR signal transduction system in A431 cells
On the basis of the above evidence showing that PGE2 transactivates EGFR and activates iNOS/GC intracellular signaling, we investigated the role of iNOS signaling in the EGFR cascade in A431 cells. Exposure to PGE2 (1 µM for 18 h) markedly stimulated iNOS activity (4-fold, P<0.01) whereas inhibition of PKA or Src activity significantly decreased it (Fig. 8 A), demonstrating that iNOS controls PGE2 effects through PKA and Src. EGF (10 ng/ml) increased NO levels >5-fold (P<0.01) while blockade of EGFR through AG1478 (1 µM) suppressed both PGE2 and EGF-induced NOS activity (Fig. 8A ). Moreover, silencing iNOS expression by si-iNOS mRNA reduced EGF-induced growth and invasion as well as ERK1/2 activation (Fig. 8B-D ), providing clear evidence that in squamous cell carcinoma the signaling activated by PGE2/EGFR transactivation impinges on the iNOS pathway to execute its functional effects.


Figure 8
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  		Figure 8. PGE2 and EGF activate iNOS activity: activation of iNOS is required for EGF-induced cell functions. A) 18 h treatment of A431 cells with PGE2 (1 µM) or EGF (10 ng/ml) induces iNOS activity. AG1478 (10 µM), PP1 (500 nM), or H89 (500 nM) (40 min) inhibit PGE2 (1 µM)-induced NOS activity. Data are reported as pmol/min/mg proteins and are the means ± SE of 3 experiments run in triplicate. **P < 0.01 vs. control response, ##P < 0.01 vs. PGE2 or EGF stimulation, #P < 0.5 vs. PGE2 alone. B) si-iNOS RNA reduced EGF-induced ERK1/2 phosphorylation, C) cell growth, and D) invasion. B) Representative Western blot showing the ERK1/2 phosphorylation induced by EGF (10 µg/ml) in the presence of si-iNOS or si-Control RNA. The gel is representative of three with similar results. Ratio = optical density of phospho-ERK1/2/total ERK1/2. C) A431 cell proliferation and D) invasion induced by EGF in the presence of si-iNOS or si-Control RNA. Data are reported as total cells counted/well for invasion or as absorbance (540 nm/well) for MTT assay; they are the means ± SE of 4 experiments run in triplicate. ***P < 0.001 vs. control response, ##P < 0.01 and ###P < 0.001 vs. EGF stimulation.

iNOS/GC pathway lies upstream to ERK1/2 activity and proliferation in growing A431 tumors
To evaluate whether the observed signaling events triggered by PGE2 via iNOS/GC pathway in vitro (i.e., ERK1/2 activity and proliferation) would operate in A431 cells grown as a tumor mass in vivo, A431 tumors were produced in nude mice. Immunohistochemical analyses showed the capability of tumor mass to produce and respond to prostanoids, since COX-2, m-PGES-1, and EP2 receptors were markedly expressed (Fig. 9 A–C). Moreover, tumor mass showed high levels of iNOS, suggesting the presence of elevated intratumoral levels of NO (Fig. 9D ).


Figure 9
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  		Figure 9. In A431 tumors, the iNOS/GC pathway lies upstream of the proliferative index and ERK1/2 phosphorylation. A–D) COX-2, m-PGES-1, EP2, and iNOS expression in A431 tumors evaluated by immunohistochemistry. Staining was performed in control A431 tumors after 14 days from implants (20x) documenting the existence of the biochemical machinery for the production/response to prostanoids and NO. E–H) Ki-67 and pERK1/2 expression in tumors evaluated by immunohistochemistry. Staining for Ki-67 and pERK1/2 was performed in control (E, G) and ODQ-treated A431 tumors (F, H). I) Western blot analysis of EGFR in tumors. The gel is representative of two gels with similar results. Ratio = optical density of phospho-tyrosine/total EGFR. Immunohistochemical pictures demonstrate marked inhibition of Ki-67 and ERK1/2 expression in treated animals (right panels) relative to controls (left panels) (40x) whereas EGFR phosphorylation was unchanged.

When tumors were well established (10 days), nude mice were treated with the GC inhibitor ODQ, and the proliferative index and ERK1/2 phosphorylation were measured (day 14) in the tumor mass. Peritumoral treatment with ODQ (10 µM/day/mice) reduced cGMP production compared with the control group (2.97±1.4 vs. 6.3±1.1 fmol/mg protein). In ODQ-treated mice, the proliferative index, measured by Ki-67 marker, and ERK1/2 phosphorylation were selectively reduced in the epithelial components of the tumor mass relative to controls (Fig. 9E-H ). We then examined whether ODQ treatment would modify EGFR activity in these tumors. Immunoprecipitation of EGFR and subsequent phosphotyrosine Western blot analysis indicated that EGFR existed in an activated state and was unaffected by cGMP inhibition (Fig. 9G ). Thus, consistent with the in vitro observations, in growing A431 tumors the iNOS/GC signaling pathway is upstream to ERK1/2 activity and affects the proliferative status of tumor cell population, whereas it lies downstream to EGFR signaling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PGE2 and EGF independently propagate through their respective membrane receptors, signals inducing the proliferation of a wide variety of epithelial tumors (7 , 8 , 45 46) . However, growing evidence indicates that the epithelial tumorigenic drive is largely sustained by the close interplay of the prostanoid with the EGF system, resulting in the transactivation of EGFR. In this study we examined the influence of the tumor-generated NO on the PGE2/EGFR intracellular signaling in squamous tumor cells, A431 and SCC-9, providing evidence that the NOS/GC pathway leads to downstream activation of ERK1/2 activation, cell invasiveness, and growth. The first clue for the NO relevance originates from the observation of enhanced iNOS activity in cultured tumor cells after their exposure to PGE2 or EGF. Another indication comes from the iNOS silencing experiments (either siRNA technology or pharmacological inhibitors), which strongly reduced the PGE2-promoted stimulation of intracellular signals (ERK1/2 phosphorylation) and tumorigenic activity (growth and invasiveness). Although not investigated in this study, downstream of GC, a mediator for MAPK signaling pathway activation, may be the protein kinase cGMP-dependent (PKG) observed earlier by us and others in different cell types (47 48 49) (see Fig. 10 ). In analogy with the cultured tumor cells, the iNOS pathway appears to be activated in vivo on proliferating epithelial tumor cells, A431, as its blockade by ODQ treatment severely curtails their proliferative activity (measured by the Ki-67) or ERK1/2 phosphorylation. The in vivo condition shown in this study illustrates the presence of the PGE2/EGFR/NOS-GC interaction paradigm. In fact, we found high expression levels of iNOS, COX-2, and m-PGES-1, suggesting the presence of elevated levels of PGE2 and NO in tumors and of both EP2 receptor subtype and phosphorylated EGFR, which are suggestive of their potential functional role in tumor progression.


Figure 10
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  		Figure 10. Schematic model of PGE2 signaling in squamous cell carcinoma. In this study we found that, in tumor cells, the PGE2/EP2 receptor system transactivates EGFR via PKA and c-Src activation. The iNOS/CG signaling pathway regulates the downstream molecular/cellular effects of PGE2-activated-EGFR, such as ERK1/2 phosphorylation, tumor cell invasion, and proliferation. In the scheme, the potential direct activation of iNOS by PGE2/EP4/Akt signaling pathway is also illustrated. AC, adenylate cyclase; PKA, protein kinase A; GC, guanylate cyclase; PKG, protein kinase G.

The issue of PGE2/EGFR transactivation (i.e., whether the prostanoid conveys its tumorigenic potential by promoting the phosphorylation of the EGFR) appears straightforward, at least for A431 and SCC-9, the epithelial tumor cells examined in this study. The phenomenon of EGFR tyrosine kinase transactivation by prostanoid G-protein-coupled receptors (GPCR) has been described for transducing mitogenic signals and tumor growth, suggesting that multiple intracellular signal pathways can be coactivated after prostanoid-receptor binding (30 31 32 , 37 , 50 , 51) . On the other hand, PGE2 has also been reported to promote tumor cell growth in an EGFR-independent manner (52) . These different signal transduction pathways induced by PGE2 seem to depend on the type of tumor cells under study and on the subtype of GPCR involved in the signaling (28 , 30 , 53) . In squamous cell carcinoma, PGE2, upon binding to its receptor subtype EP2, initiates a cascade of events involving both PKA and cSrc, which culminates with the phosphorylation of EGFR. Previous reports have also described the transactivation of EGFR mediated by EP2 receptor in human Ishikawa endometrial adenocarcinoma cells or EP4 receptor in human colorectal cells (31 , 32 , 54) . PGE2-mediated effects on tumor and host cells depend on the expression of four different EP receptors (EP1-EP4), providing intracellular signaling by different mechanisms [e.g., increase of Ca2+ and inositol levels (EP1), elevation of cAMP (EP2/EP4), activation of PI3K-Akt signal transduction pathway (EP4), or reduction of cAMP synthesis (EP3)] (20) . Although A431 cells express all four receptor subtypes (EP1 through EP4), only the EP2 subtype appears to be exclusively endowed with the ability to transduce the PGE2 signal, since its selective stimulation via Butaprost stimulates ERK1/2 phosphorylation, cell invasiveness, and growth, all of which are sensitive to EGFR inhibition. As a corollary to the PGE2/EP2-promoted transactivation of EGFR, analysis of signals such as PKA and c-Src (31 , 32 , 54) provides insight into their role on the mechanism of EGFR transactivation in squamous carcinoma cells. In fact, the evidence obtained in cultured cells with the specific inhibitors of cSrc or PKA, PP1, and H89, respectively (for the latter, also by silencing its mRNA) (55) , provides compelling evidence that these intermediate molecules are necessary for EGFR activation. Moreover, these inhibitors as well as the blockade of EGFR phosphorylation by AG1478 obliterate the PGE2 tumor-promoting activity as well as iNOS activity. Silencing of iNOS also suppresses EGF-induced ERK1/2 activation and tumor cell growth and invasion, providing clear evidence that iNOS signal transduction pathway is a molecular mechanism required for the PGE2/EP2-EGFR-induced postreceptor effects in A431 cells. These results do not exclude the possibility that PGE2 may directly activate iNOS without EGFR. For example, the prostanoid may activate Akt by stimulating the EP4 subtype receptor, which induces phosphorylation of iNOS (56 , 57) .

In conclusion, the principal finding of this study is that, in squamous carcinoma cell lines A431 and SCC-9, iNOS is the dominant downstream player in the control of PGE2-induced growth and invasion, being instrumental for the prevailing tumorigenic drive (i.e., the EGF-EGFR system). In other epithelial tumors, such as NSCLC, PGE2 exerts its tumor growth potential independently from the EGF-EGFR system (51) . On the other hand, the role of NOS as a crucial intermediate in the progression of squamous carcinomas finds a clinical correlate in head and neck cancer, in which specimens taken from patients have been described to express high levels of NOS, which correlated strongly with tumor aggressiveness (3) .

The recognition that iNOS/GC signaling works in concert with the stromal-derived PGE2 and the EGF protumorigenic pathway reinforces the key role exerted by NOS in inflammation and cancer, and the link between the two pathologies. The enrichment of inflammatory cells in the microenvironment around tumors provides an optimal milieu for cell proliferation, invasion, and angiogenesis, thereby favoring tumor development.


   ACKNOWLEDGMENTS
 
Helpful discussions with Prof. A. Giachetti (Lifetech Srl, Florence, Italy) are greatly acknowledged. This work was supported by Italian Ministry for Research (FIRB project RBNE01A882_002 and PRIN project 2004065317_001) and the EU project EICOSANOX FP6 funding (LSHM-CT-2004–0050333). This publication reflects only the authors’ views. The European Commission is not liable for any use that may be made of information herein. S.D. was supported by funds from NuGO (FOOD-CT-2004–506360). F.F. was a recipient of a fellowship from Fondazione Callerio (Trieste, Italy).


   FOOTNOTES
 
1 These authors have contributed equally to this work.

Received for publication November 13, 2006. Accepted for publication February 15, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Shah, J. P., Medina, J. E., Shaha, A. R., Schantz, S. P., Marti, J. R. (1993) Cervical lymph node metastasis. Curr. Probl. Surg. 30,1-335[Medline]
  2. Folkman, J. (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1,27-31[CrossRef][Medline]
  3. Gallo, O., Masini, E., Morbidelli, L., Franchi, A., Fini-Storchi, I., Vergari, W. A., Ziche, M. (1998) Role of nitric oxide in angiogenesis and tumor progression in head and neck cancer. J. Natl. Cancer Inst. 90,587-596[Abstract/Free Full Text]
  4. Jenkins, D. C., Charles, I. G., Thomsen, L. L., Moss, D. W., Holmes, L. S., Baylis, S. A., Rhodes, P., Westmore, K., Emso, P. C., Moncada, S. (1995) Role of nitric oxide in tumor growth. Proc. Natl. Acad. Sci. U. S. A. 92,4392-4396[Abstract/Free Full Text]
  5. Thomsen, L. L., Miles, D. W., Happerfield, L., Bobrow, L. G., Knowles, R. G., Moncada, S. (1995) Nitric oxide synthase activity in human breast cancer. Br. J. Cancer 72,41-44[Medline]
  6. Xu, W., Liu, L. Z., Loizidou, M., Ahmed, M., Charles, I. G. (2002) The role of nitric oxide in cancer. Cell. Res. 12,311-320[CrossRef][Medline]
  7. Dubois, R. N., Abramson, S. B., Crofford, L., Gupta, R. A., Simon, L. S., Vande Putte, L. B., Lipsky, P. E. (1998) Cyclooxygenase in biology and disease. FASEB J. 12,1063-1073[Abstract/Free Full Text]
  8. Cao, Y., Prescott, S. M. (2002) Many actions of Cyclooxygenase-2 in cellular dynamics and in cancer. J. Cell. Physiol. 190,279-286[CrossRef][Medline]
  9. Park, S. W., Lee, S. L., Song, S. H., Heo, D. S., Park, B. J., Lee, D. W., Kim, K. T., Sung, M. W. (2003) The effect of nitric oxide on cyclooxygenase-2 (COX-2) overexpression in head and neck cancer cell lines. Int. J. Cancer 107,729-738[CrossRef][Medline]
  10. Wendum, D., Masliah, J., Trugnan, G., Fléjou, J. F. (2004) Cyclooxygenase-2 and its role in colorectal cancer development. Virchows Arch. 445,327-333[CrossRef][Medline]
  11. Denkert, C., Kobel, M., Pest, S., Koch, I., Berger, S., Schwabe, M., Siegert, A., Reles, A., Klosterhalfen, B., Hauptmann, S. (2002) Expression of cyclooxygenase 2 is an independent prognostic factor in human ovarian cancer. Am. J. Pathol. 160,893-903[Abstract/Free Full Text]
  12. Bae, S. H., Jung, E. S., Park, Y. M., Kim, B. S., Kim, B. K., Kim, D. G., Ryu, W. S. (2001) Expression of cyclooxygenase-2 (COX-2) in hepatocelluler carcinoma and growth inhibition of hepatoma cell lines by a COX-2 inhibitor, NS-398. Clin. Cancer Res. 7,1410-1418[Abstract/Free Full Text]
  13. Tsujii, M., Kawano, S., DuBois, R. N. (1997) Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc. Natl. Acad. Sci. U. S. A. 94,3336-3340[Abstract/Free Full Text]
  14. Salvemini, D., Misko, T. P., Masferrer, J. L., Seibert, K., Currie, M. G., Needleman, P. (1993) Nitric oxide activates cyclooxygenase enzymes. Proc. Natl. Acad. Sci. U. S. A. 90,7240-7244[Abstract/Free Full Text]
  15. Salvemini, D., Currie, M. G., Mollace, V. (1996) Nitric oxide-mediated cyclooxygenase activation. A key event in the antiplatelet effect of nitrosovasodilatators. J. Clin. Invest. 97,2562-2568[Medline]
  16. Timoshenko, A. V., Lala, P. K., Chakraborty, C. (2004) PGE2-mediated upregulation of iNOS in murine breast cancer cells through the activation of EP4 receptors. Int. J. Cancer 108,384-389[CrossRef][Medline]
  17. Chang, S. H., Liu, C. H., Conway, R., Han, D. K., Nithipatikum, K., Trifan, O. C., Lane, T. F., Hla, T. (2004) Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression. Proc. Natl. Acad. Sci. U. S. A. 10,591-596
  18. Gupta, R. A., Dubois, R. N. (2001) Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2. Nat. Rev. Cancer 1,11-21[CrossRef][Medline]
  19. Sheng, H., Shao, J., Washington, M. K., DuBois, R. N. (2001) Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J. Biol. Chem. 276,18075-18081[Abstract/Free Full Text]
  20. Hata, A. N., Breyer, R. M. (2004) Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation. Pharmacol. Ther. 103,147-166[CrossRef][Medline]
  21. Regan, J. W. (2003) EP2 and EP4 prostanoid receptor signaling. Life Sci. 74,143-153[CrossRef][Medline]
  22. Breyer, R. M., Bagdassarian, C. K., Myers, S. A., Breyer, M. D. (2001) Prostanoid receptors: subtypes and signalling. Annu. Rev. Pharmacol. Toxicol. 41,561-690
  23. Funk, C. D., Furci, L., Fitzgerald, G. A., Grygorczyk, R., Rochette, C., Bayne, M. A., Abramovitz, M., Adam, M., Metters, K. M. (1993) Cloning and expression of a cDNA for the human prostaglandin E receptor EP1 subtype. J. Biol. Chem. 268,26767-26772[Abstract/Free Full Text]
  24. Regan, J. W., Bailey, T. J., Pepperl, D. J., Pierce, K. L., Bogardus, A. M., Donello, J. E., Fairbairn, C. E., Kedezie, K. M., Woodward, D. F., Gil, D. W. (1994) Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP2 subtype. Mol. Pharmacol. 46,213-220[Abstract]
  25. Sugimoto, Y., Namba, T., Honda, A., Hayashi, Y., Negishi, M., Ichikawa, A., Narumiya, S. (1992) Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. J. Biol. Chem. 267,6463-6466[Abstract/Free Full Text]
  26. Honda, A., Sugimoto, Y., Namba, T., Watabe, A., Irie, A., Negishi, M., Narumiya, S., Ichikawa, A. (1993) Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype. J. Biol. Chem. 268,7759-7762[Abstract/Free Full Text]
  27. Timoshenko, A. V., Guoxiong, X., Chakrabarti, S., Lala, P. K., Chakraborty, C. (2003) Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells. Exp. Cell Res. 289,265-274[CrossRef][Medline]
  28. Fujino, H., Regan, J. W. (2003) Prostanoid receptors and phosphatidylinositol 3-kinase:a pathway to cancer. Trends Pharmacol. Sci. 24,335-340[CrossRef][Medline]
  29. Majima, M., Amano, H., Hayashi, I. (2003) Prostanoid receptor signaling relevant to tumor growth and angiogenesis. Trends Pharmacol. Sci. 24,524-529[CrossRef][Medline]
  30. Pai, R., Soreghan, B., Szabo, I. L., Pavelka, M., Baatar, D., Tarnavski, A. J. (2002) Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy. Nat. Med. 8,289-293[CrossRef][Medline]
  31. Buchanan, F. G., Wang, D., Bargiacchi, F., DuBois, R. N. (2003) Prostaglandin E2 regulates cell migration via the intracellular activation of the epidermal growth factor receptor. J. Biol. Chem. 278,35451-35457[Abstract/Free Full Text]
  32. Sales, K. J., Maudsley, S., Jabbour, N. H. (2005) Elevated Prostaglandin EP2 receptor in endometrial adenocarcinoma cells promotes vascular endothelial growth factor expression via cyclic 3',5'-adenosin monophosphate-mediated transactivation of the epidermal growth factor receptor and extracellular signal-regulated kinase1/2 signaling pathways. Mol. Endocrinol. 18,1533-1545[CrossRef]
  33. Gullick, W. J. (2001) The Type 1 growth factor receptors and their ligands considered as a complex system. Endocr. Relat. Cancer 8,75-82[Abstract]
  34. Yarden, Y. (2001) The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur. J. Cancer 37 S4,S3-S8
  35. Lui, V. W., Grandis, J. R. (2002) EGFR-mediated cell cycle regulation. Anticancer Res. 22,1-11[Medline]
  36. Gallo, O., Fabbroni, V., Sardi, I., Magnelli, L., Boddi, V., Franchi, A. (2002) Correlation between nitric oxide and cyclooxygenase-2 pathways in head and neck squamous cell carcinoma. Biochem. Biophys. Res. Commun. 299,517-524[CrossRef][Medline]
  37. Chen, Z. G., Zhang, X., Li, M., Wang, Z., Wieand, H. S., Grandis, J. R., Shin, D. M. (2004) Simultaneously targeting epidermal growth factor receptor tyrosine kinase and cyclooxygenase-2, an efficient approach to inhibition of squamous cell carcinoma of head and neck. Clin. Cancer Res. 10,5930-5939[Abstract/Free Full Text]
  38. Taraboletti, G., Morbidelli, L., Donnini, S., Parenti, A., Granger, H. J., Giavazzi, R., Ziche, M. (2000) The heparin binding 25 kDa fragment of thrombospondin-1 promotes angiogenesis and modulates gelatinase and TIMP-2 production in endothelial cells. FASEB J. 14,1674-1676[Free Full Text]
  39. Donnini, S., Morbidelli, L., Taraboletti, G., Ziche, M. (2004) ERK1/2 and p38 MAPK regulate MMP/TIMP balance and function in response to thrombospondin-1 fragments in the microvascular endothelium. Life Sci. 74,975-985
  40. Cantara, S., Donnini, S., Morbidelli, L., Giachetti, A., Schulz, R., Memo, M., Ziche, M. (2004) Physiological levels of amyloid peptides stimulate the angiogenic response through FGF-2. FASEB J. 18,943-945
  41. Elbashir, S. M., Harborth, J., Bechert, K., Tuschl, T., Weber, K. (2001) Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell. Sci. 114,4557-4565[Medline]
  42. Parenti, A., Morbidelli, L., Cui, X. L., Douglas, J. G., Hood, J. D., Granger, H. J., Ledda, F., Ziche, M. (1998) Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal regulated kinase1/2 activation in postcapillary endothelium. J. Biol. Chem. 273,4220-4226[Abstract/Free Full Text]
  43. Fujino, H., Salvi, S., Regan, J. W. (2005) Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. Mol. Pharmacol. 68,251-259[Abstract/Free Full Text]
  44. Galisteo, M., Marc, N., Fautrel, A., Guillouzo, A., Corcos, L., Lagadic-Gossmann, D. (1999) Involvement of cyclic nucleotide- and calcium-regulated pathways in phenobarbital-induced cytochrome P-450 3A expression in mouse primary hepatocytes. J. Pharmacol. Exp. Ther. 290,1270-1277[Abstract/Free Full Text]
  45. Normanno, N., De Luca, A., Bianco, C., Strizzi, L., Mancino, M., Macello, M. R., Carotenuto, A., De Feo, G., Caponigro, F., Salomon, D. S. (2006) Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366,2-16[CrossRef][Medline]
  46. Jorissen, R. N., Walker, F., Pouliot, N., Garret, T. P. J., Ward, C. W., Burgess, A. W. (2003) Epidermal growth factor receptor: mechanism of activation and signalling. Exp. Cell Res. 284,31-53[CrossRef][Medline]
  47. Hood, J., Granger, H. J. (1998) Protein kinase G mediates vascular endothelial growth factor-induced Raf-1 activation and proliferation in human endothelial cells. J. Biol. Chem. 273,23504-23508[Abstract/Free Full Text]
  48. Jadeski, L. C., Chakraborty, C., Lala, P. K. (2003) Nitric oxide-mediated promotion of mammary tumor cell migration requires sequential activation of nitric oxide synthase, guanylate cyclase and mitogen activated protein kinase. Int. J. Cancer 106,496-504[CrossRef][Medline]
  49. Hood, J. D., Meininger, C. J., Ziche, M., Granger, H. J. (1999) VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am. J. Physiol. 274,H1054-H1058
  50. Shao, J., Evers, M., Sheng, H. (2004) Prostaglandin E2 synergistically enhances receptor tyrosine kinase-dependent signaling system in colon cancer cells. J. Biol. Chem. 279,14287-14293[Abstract/Free Full Text]
  51. Thomas, S. M., Bhola, N. E., Zhang, Q., Contrucci, S. C., Wentzel, A. L., Freilino, M. L., Gooding, W. E., Siegfried, J. M., Chan, D. C., Grandis, J. R. (2006) Cross-talk between G protein-coupled receptor and epidermal growth factor receptor signaling pathways contributes to growth and invasion of head and neck squamous cell carcinoma. Cancer Res. 66,11831-1131[Abstract/Free Full Text]
  52. Krysan, K., Reckamp, K. L., Dalwadi, H., Sharma, S., Rozengurt, E., Dohadwala, M., Dubinett, S. M. (2005) Prostaglandin E2 activates mitogen-activated protein kinase/Erk pathway signalling and cell proliferation in non-small cell lung cancer cells in an epidermal growth factor receptor-independent manner. Cancer Res. 65,6275-6280[Abstract/Free Full Text]
  53. Pai, R., Nakamura, T., Moon, W. S., Tarnawski, A. S. (2003) Prostaglandins promote colon cancer cell invasion; signalling by cross-talk between two distinct growth factor receptors. FASEB J. 17,1640-1647[Abstract/Free Full Text]
  54. Mutoh, M., Watanabe, K., Kitamura, T., Shoji, Y., Takahashi, M., Kawamori, T., Tani, K., Kobayashi, M., Maruyama, T., Kobayashi, K., et al (2002) Involvement of prostaglandin E receptor subtype EP4 in colon carcinogenesis. Cancer Res. 62,28-32[Abstract/Free Full Text]
  55. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., Idaka, H. (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. 265,5267-5272[Abstract/Free Full Text]
  56. Fujino, H., West, K. A., Regan, J. W. (2002) Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. J. Biol. Chem. 277,2614-2619[Abstract/Free Full Text]
  57. Iwakiri, Y., Tsai, M. H., McCabe, T. J., Gratton, J. P., Fulton, D., Groszmann, R. J., Sessa, W. C. (2002) Phosphorylation of eNOS initiates excessive NO production in early phases of portal hypertension. Am. J. Physiol. 282,H2084-H2090



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