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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Cyclooxygenase (COX)-2 is known to correlate with poor cancer prognosis and to contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal prostaglandin E2 (PGE2)–prostaglandin E2 receptor (EP)3 signaling appears critical for tumor-associated angiogenesis and tumor growth. Here we tested whether the EP3 receptor has a critical role in tumor metastasis. Lewis lung carcinoma (LLC) cells were intravenously injected into WT mice and mice treated with the COX-2 inhibitor NS-398. The nonselective COX inhibitor aspirin reduced lung metastasis, but the COX-1 inhibitor SC560 did not. The expression of matrix metalloproteinases (MMP)-9 and vascular endothelial growth factor (VEGF)-A was suppressed in NS-398-treated mice compared with PBS-treated mice. Lungs containing LLC colonies were markedly reduced in EP3 receptor knockout (EP3−/−) mice compared with WT mice. The expression of MMP-9 and VEGF-A was downregulated in metastatic lungs of EP3−/− mice. An immunohistochemical study revealed that MMP-9-expressing endothelial cells were markedly reduced in EP3−/− mice compared with WT mice. When HUVEC were treated with agonists for EP1, EP2, EP3, or EP4, only the EP3 agonist enhanced MMP-9 expression. These results suggested that EP3 receptor signaling on endothelial cells is essential for the MMP-9 upregulation that enhances tumor metastasis and angiogenesis. An EP3 receptor antagonist may be useful to protect against tumor metastasis. (Cancer Sci 2009; 100: 2318–2324)

Metastasis is the primary cause of mortality in cancer patients.(1) While it has been recognized that the movement of neoplastic cells is not a random process, the molecular and cellular mechanisms governing their movement, survival through foreign tissue, and parameters for selection of their final destination remain unclear. To produce clinically relevant lesions, metastatic cells must complete the following steps, involving: the ability of tumor cells to escape from their original position, to attach to the extracellular matrix (ECM), to degrade the ECM component, and to migrate through these ECM. Thus, both cell–cell adhesion and ECM degradation represent significant barriers to the metastasis of tumor cells.(2)

Nonsteroidal anti-inflammatory drugs (NSAID), which block the enzyme activity of cyclooxygenase (COX), have been widely used for anti-inflammatory and analgesic purposes. Several papers have reported that a significant reduction in mortality from colorectal cancer occurred depending on the cumulative doses of an NSAID, but on the other hand further evidence suggests that NSAID also affect the incidence and progression of other types of cancer, pointing to a possible role of COX in other types of tumor formation.(3–5)

Prostaglandins comprise a large family of small lipid molecules derived from COX-1- and COX-2-mediated metabolism of arachidonic acid to prostaglandin G2 (PGG2)/prostaglandin H2(H2).(6) Cell-specific prostaglandin synthases convert prostaglandin H2 (PGH2) into a series of bioactive prostaglandins, including prostaglandin E2 (PGE2). Reported clinical and experimental data indicate that PGE2 can enhance the invasiveness of colon cancer cells and the tumorigenic potential of intestinal epithelial cells.(7–9) Four PGE2 subtype receptors (prostaglandin E2 receptor [EP]1, EP2, EP3, and EP4 receptors) have been cloned and characterized. Furthermore, with the use of the cloned receptors, agonists and antagonists highly selective for each of the four subtypes have been or are in the process of being developed.(10)

We have previously reported that host stromal PGE2–EP3 signaling is critical for tumor-associated angiogenesis and tumor growth.(11) Thus, we hypothesized that PGE2–EP3 signaling may facilitate tumor metastasis. It has been reported that the expression of matrix metalloproteinases (MMP) is upregulated in advanced tumors, and a number of studies have outlined the roles of these enzymes in multiple stages of tumorigenesis, including angiogensesis, invasion, migration, and metastasis. Several MMP, including the gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B), are expressed at high levels in tumors, and in various organs and species both serum and tissue levels of MMP-9 have been well correlated with the grade of malignancy.(12) PGE2 has been reported to increase the levels of some MMP in several types of cells.(13,14) Thus, in the present study, we tested whether or not the signaling of the EP3 receptor, which is expressed at the metastatic sites, is relevant to lungs containing Lewis lung carcinoma colonies, using EP3−/− mice. We showed that EP3 signaling is required for tumor metastasis and invasion, and that this receptor signaling upregulates the expression of MMP-9 in endothelial cells. The blockade of EP3 receptor has promise as a contributory process in the inhibition of tumor metastasis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Drugs.  Aspirin (10 mg/mL) was provided by Merck (Drivehouse, NJ, USA), NS-398 (3 mg/mL) was obtained from Cayman Chemicals (Ann Arbor, MI, USA), and SC-560 (3 mg/mL) was provided by Pfizer (New York, NY, USA). These drugs were suspended in 5% gum arabic in physiological saline and administered orally in a suspension (0.1 mL/10 mg of body mass) twice a day (every 12 h) beginning on the day of cell implantation. Ten nM of the EP receptor-selective agonists ONO-DI-004 (EP1), ONO-AEI-257 (EP2), ONO-AE-248 (EP3), and ONO-AEI-329 (EP4), which were developed by us previously, were added to HUVEC.(15)

Cell lines.  HUVEC purchased from Kurabo (Tokyo, Japan) were cultured in 10% FBS and endothelial cell growth supplement (EGM-2 MV; Cambrex Bioscience, Wekerville, MD, USA). LLC, which were originally isolated from C57Bl/6 mice, were cultured at 37°C in RPMI-1640 with 10% FBS in a humidified atmosphere containing 5% CO2. Confluent HUVEC incubated in serum-free media were treated with 10 nm of selective EP1–4 receptor agonists and PBS for 24 h.

RT-PCR analysis in HUVEC.  After 24 h of treatment with EP receptor agonists at a final concentration of 0.1 nM, HUVEC were collected and homogenized with Trizol (Invitrogen, Carlsbad, CA, USA). Extracted RNA was converted to cDNA for RT-PCR analysis for MMP-9 and GAPDH. The following primers were used: 5′-GAGACCGGTGAGCTGATAG-3′ (sense) and 5′-TCGAAGATGAAGGGGAAGTG-3′ (antisense) for MMP-9, and 5′-GAAGGTGAAGGTCGGACTC-3′ (sense) and 5′-GAAGATGGTGATGGGATTTC-3′ (antisense) for human GAPDH.

Animals.  Male C57Bl/6 mice (6–8 weeks old), weighing 25–30 g were obtained from CLEA Japan Int Shizuoka Laboratory Animal Center (Fuji, Japan). EP3 receptor knockout mice (EP3−/−, male, 8 weeks old), all with a C57Bl/6 hybrid background, were developed in our lab.(13) They were maintained at a constant humidity (60 ± 5%) and temperature (20 ± 1°C) and kept continuously on a 12:12 h L:D cycle. All animals were provided with food and water ad libitum. All experiments were carried out in accordance with the guidelines for animal experiments of Kitasato University School of Medicine.

Lung metastasis model.  LLC cells were harvested and washed three times with PBS. The cells were suspended in PBS at a density of 5 × 106 cells/mL, and 100 μL of the resulting suspension was injected into the tail vein. On day 28, C57Bl/6 and EP3−/− mice injected with LLC were killed with an excess dose of ether, and the lungs were then surgically resected. The isolated lungs were fixed with Bouin’s solution. Then, the number of metastatic colonies was counted under a light microscope.

Immunohistochemical studies.  Lung tissue was immediately fixed with 4% paraformaldehyde in 0.1 m phosphate buffer solution (pH 7.4). After fixation, the tissue was dehydrated with a graded series of ethanol solutions, and then embedded in paraffin. Each section (4 μm) of the paraffin-embedded tissue was mounted on glass slide, deparaffinized with xylene, and then placed in cold (4°C) acetone for immunostaining.

The procedure for staining dehydrated sections using a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) was as follows: (1) incubation with diluted normal horse serum; (2) incubation with the diluted (1/500) MMP-9 polyclonal antibody; (3) incubation with biotinylated anti-IgG; (4) incubation with the avidin–biotin–peroxidase complex; (5) placement in 0.02% 3,3′-diaminobenzine (DAB) and 0.3% nickel ammonium sulfate in 50 mm Tris-HCl buffer (pH 7.4); (6) color development by immersion in DAB solution containing 0.005% H2O2; and (7) examination and photomicrography with a light microscope. Using the same procedures, we stained immunoreactive VEGF and CD31, as we have previously reported.(11,16,17)

RT-PCR analysis in lung tissues.  The lungs were homogenized with Trizol, and RNA was extracted and converted to cDNA using RT-PCR analysis for VEGF-A, CD31, and GAPDH. Mice were injected with LLC and harvesting was undertaken 7 days afterwards. The primers were as follows: 5′-CTGTGCAGGCTGCTGGTAACGATGAAGC-3′ (sense) and 5′-CCGGTGAGAGGTCTGGTTCCCGAAACC-3′ (antisense) for VEGF-A; 5′-AGCTAGCAAGAAGCAGGAAGGA-3′ (sense) and 5′-GTAATGGTGTTGGCTTCCACA-3′ (antisense) for CD31; 5′-GAGTATGTCGTGGAGTCTACTG-3′ (sense) and 5′-GATGCAGGGATGATGTTCTG-3′ (antisense) for GAPDH.

Determination of MMP-9 and MMP-2 mRNA levels in lung tissues by real-time PCR.  Transcripts encoding MMP-9, MMP-2, and GAPDH were quantified by real-time RT-PCR analysis. The expression of each mRNA was qualified at defined time after LLC injection. Total RNA from lung tissue was extracted with TRIzol reagent (Invitrogen). The amount of RNA was measured by BioPhotometer (Eppendorf, Tokyo, Japan). Subsequently, synthesis of first-strand cDNA from total RNA was carried out with 1 μg of total RNA, 200 U of ReverTra Ace (reverse transcriptase; Toyobo, Osaka, Japan), 40 nmol dNTP mixture (Toyobo), 20 U RNase inhibitor (Toyobo), and 20 pmol oligo(dT)20 in a total volume of 40 μL. The reactions were incubated initially at 30°C for 10 min and then at 42°C for 40 min, followed by inactivation of the reaction at 99°C for 5 min. The real-time PCR primers were designed using Primer3 software (http://primer3.sourceforge.net/) on the basis of the GenBank data. The following primers were used for real-time PCR:5′-CCCATGTCACTTTCCCTTCAC-3′ (sense) and 5′-GCCGTCCTTATCGTAGTCAGC-3′ (antisense) for MMP-9; 5′-TTCAAGGACCGGTTTATTTGG-3′ (sense) and 5′-CACAGCGTCAATCTTTTCTGG-3′ (antisense) for MMP-2; and 5′-ACATCAAGAAGGTGGTGAAGC-3′ (sense) and 5′-AAGGTGGAAGAGTGGGAGTTG-3′ (antisense) for GAPDH (Applied Sigma-Aldrich, Tokyo, Japan).

Gene-specific primers and cDNA solution were added to SYBR Premix Ex Taq II (Takara, Otsu, Japan) and subjected to PCR amplification in the CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The reaction were incubated initially at 95°C for 10 sec as initial denaturation step, 40-cycle shuttle PCR; at 95°C for 3 sec as denaturation step and at 60°C for 20 sec as annealing and extension steps. The obtained threshold cycle (Ct) values were processed for further calculations according to the comparative Ct method. Expression levels of target genes were normalized to the housekeeping gene GAPDH, giving the ΔCt value. Finally, the gene expression level was calculated as inline image, giving the final value that was normalized to GAPDH.

ELISA analysis.  The lung tissue was removed from the mice on day 14 and homogenized immediately with 2 mL PBS. The supernatants (1500g, 15 min, 4°C) and mouse pro-MMP-9 and VEGF-A were measured using ELISA (R&D Systems, Minneapolis, MN, USA) and normalized to the total protein concentration, determined by BCA assay (Pierce, Rockford, IL, USA), as reported previously.(18)

Statistical analysis.  Data is expressed as mean ± SD. Comparisons among multiple groups were carried out by analysis of variance (ANOVA). Comparisons between the two groups were made using Student’s t-test. A P-value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

COX-2 requirement for forming metastatic colonies.  The effects of COX inhibitors on tumor metastasis were tested using LLC cells, which were syngeneic for C57Bl/6 mice (Fig. 1). In control C57Bl/6 mice treated with PBS, the number of colonies formed in the lungs was 37.1 ± 12.5. Daily oral administration of SC560, selectively acting on COX-1, had no significant effect (28.8 ± 10.7). In contrast, the COX-2-selective inhibitor, NS-398, significantly reduced the number of colonies, as did aspirin and the non-selective COX inhibitor (NS-398, 8.8 ± 3.8; aspirin, 13.6 ± 4.5). This result suggested that COX-2 is a major determinant for forming LLC colonies in this model.

image

Figure 1.  Effect of COX-2 inhibitors on tumor metastasis. A suspension of Lewis lung carcinoma cells that were syngeneic for C57Bl/6 was injected into the tail vein of wild-type mice. COX-2 inhibitors (NS-398, 3 mg/mL; aspirin, 10 mg/mL) were administered orally in the suspension (0.1 mL/10 g of body mass) twice a day (every 12 h) beginning on the day of cell injection and continuing throughout the 28-day experimental period. Data are means ± SD for the number of mice (all n = 6/group). *P < 0.05 versus vehicle-treated mice (ANOVA).

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Reduced MMP-9 and VEGF-A expression in NS-398-treated mice.  To determine whether NS-398 affects tumor metastasis formation, pro-MMP-9 in the lung tissue was measured. The protein level of pro-MMP-9 expression in lungs containing LLC colonies was markedly reduced in NS-398-treated mice compared with that in vehicle mice (Fig. 2a; vehicle, 38.2 ± 7.1; NS-398, 4.4 ± 1.3 ng/mg protein; < 0.05). In addition, VEGF-A expression in lungs containing LLC colonies was markedly reduced in vehicle mice compared with that in NS-398-treated mice (Fig. 2b; vehicle, 68.6 ± 19.5; NS-398, 16.4 ± 6.9 ng/mg protein; < 0.05). These results suggested that the effects of COX-2 inhibitors on tumor metastasis were associated with the level of pro-MMP-9 and VEGF-A levels.

image

Figure 2.  Reduced pro-MMP-9 and vascular endothelial growth factor (VEGF)-A expression in COX-2 inhibitor-treated mice. (a) Pro-MMP-9 levels and (b) VEGF-A in lung tissue 14 days after injection of Lewis lung carcinoma cells determined by ELISA. Data are means ± SD for the number of animals (all n = 5/group). *P < 0.05 versus wild-type mice (Student’s t-test).

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Reduced LLC colony formation in EP3−/− mice.  We have previously reported that PGE2–EP3 signaling is essential for tumor growth. Seeking to identify the roles of EP3 in tumor metastasis, we compared the numbers of colonies in EP3 receptor knockout mice after intravenous injection of LLC cells with those in WT counterpart mice (Fig. 3). There were significantly fewer colonies formed in EP3−/− mice than in WT mice (by 50.5%, P < 0.05). This data suggested that EP3 signaling facilitates LLC colony formation.

image

Figure 3.  Reduced Lewis lung carcinoma colony formation in prostaglandin E2 receptor (EP)3−/− mice. (a) The number of colonies in the lungs 28 days after intravenous injection of Lewis lung carcinoma cells. EP3+/+, wild-type mice; EP3−/−, EP3 receptor knockout mice. Data are means ± SD for the number of mice (all n = 10/group). P < 0.05 versus wild-type mice (Student’s t-test). (b) Typical hematoxylin–eosin staining of metastatic lung tumors from EP3+/+ and EP3−/− mice 28 days after injection of Lewis lung carcinoma cells.

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Effect of EP3 signaling on MMP-9 expression in cultured HUVEC.  To show that PGE2 signaling via the EP3 receptor is present on the endothelial cells and is involved in tumor metastasis through the secretion of MMP-9, we examined the expression of MMP-9 mRNA in HUVEC 24 h after treatment with selective agonists for EP1, EP2, EP3, and EP4. RT-PCR analysis showed that the EP3 agonist, more than any other EP agonist, upregulated MMP-9 expression (Fig. 4a). This result suggested that the EP3 receptor enhances MMP-9 expression and promotes conditions conducive to metastasis.

image

Figure 4.  Reduced expression of MMP-9 in tumor metastasis lesions in EP3−/− mice. (a) The expression of MMP-9 in HUVEC 24 h after treatment with selective agonists (final concentration, 0.1 nM) selective for EP1, EP2, EP3, and EP4. (b) Pro-MMP-9 levels in lung tissue 14 days after injection of Lewis lung carcinoma cells determined by ELISA. Data are means ± SD for the number of animals (all n = 5/group). *P < 0.05 versus wild-type mice (Student’s t-test). (c) Determination of MMP-9 and MMP-2 mRNA levels in lung tissues by real-time PCR. Lung tissue was isolated 7, 14 days after injection of Lewis lung carcinoma. Real-time PCR was carried out. Results were compared with WT and EP3−/− mice. Data are means ± SD for the number of animals (7–14 animals). *P < 0.05 versus WT mice (Student’s t-test). (d) Immunohistochemical localization of CD31 and MMP-9 in EP3+/+. Blue arrows indicate CD31- and MMP-9-positive endothelial cells. T, tumor. (e) Immunohistochemical localization of MMP-9 in EP3+/+ and EP3−/−. Yellow arrows indicate endothelial cells and blue arrows indicated macrophage-like interstitial cells.

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Expression of MMP-9 in lungs containing LLC colonies.  Extensive experimental data obtained with ELISA and real-time PCR analysis suggested that MMP-9 significantly contributes to cancer progression (Fig. 4b,c). We found that pro-MMP-9 levels in lungs containing LLC colonies were significantly reduced in EP3−/− mice compared with WT mice (WT, 36.6 ± 5.5; EP3−/−; 4.7 ± 3.5 ng/mg protein, P < 0.05). In addition, MMP-9 mRNA expression in lung tissues was also reduced in EP3−/− mice at 7 and 14 days after LLC injection. In contrast, there was no significant difference in MMP-2 mRNA expression between WT mice and EP3−/− mice. These results indicated that EP3 signaling facilitates LLC colony formation and upregulates MMP-9 expression.

Immunohistochemical analysis of MMP-9 in lungs containing LLC colonies.  To corroborate the interaction between tumor and endothelial cells, we examined MMP-9 expression in tumors and endothelial cells by immunohistochemical analysis in the present syngeneic models. Intense staining for MMP-9 was apparent in endothelial cells near the metastatic locus in WT mice (Fig. 4d, red arrows in the left panel), whereas staining in EP3−/− mice was less marked (Fig. 4d, right panel). In WT mice, MMP-9 was also observable in macrophage-like interstitial cells present around the metastatic lesions (Fig. 4e, yellow arrows). The number of MMP-9-positive endothelial cells was markedly reduced in EP3−/− mice, whereas that of macrophage-like interstitial cells was not reduced in EP3−/− mice, suggesting that endothelial MMP-9 expression was selectively upregulated by EP3 signaling.

Reduced expression of VEGF-A in lungs containing LLC colonies in EP3−/− mice.  VEGF-A expression in lungs containing LLC colonies was markedly reduced in EP3−/− mice compared with that in WT mice, as shown by the VEGF-A protein level estimated by ELISA (Fig. 5a; WT, 92.0 ± 13.7 ng/mg protein; EP3–/–, 46.1 ± 9.2 ng/mg protein; < 0.05). In addition, VEGF-A mRNA levels were also reduced in EP3−/− mice compared with those in WT mice, determined by RT-PCR (Fig. 5b). This reduction was accompanied by reduced expression of CD31 (Fig. 5b). These results indicated that EP3 signaling upregulates VEGF-A expression with a concomitant increase in lungs containing LLC colonies.

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Figure 5.  Reduced expression of VEGF-A in tumor metastasis lesions in EP3−/− mice. (a) VEGF levels in the lungs homogenized with PBS. The levels were determined by ELISA. Data are means ± SD for the number of animals (all n = 5/group). *P < 0.05 versus wild-type mice (Student’s t-test). (b) The expression of VEGF-A and CD31 mRNA in lung tissues. Lung tissue was isolated 2 weeks after injection of Lewis lung carcinoma. (c) Immunohistochemical localization of VEGF-A and CD31 in EP3+/+ and EP3−/−. Blue arrows indicate VEGF-positive endothelial cells near the metastatic locus, black circles indicate VEGF-A-positive stromal cells around tumor cells, and red arrows indicate CD31-positive cells. T, tumor. (d) Immunohistochemical localization of CD31 and MMP-9 in EP3+/+. The localization of CD31-positive cells (red arrows in the left panel) is coincident with that of MMP-9-positive cells in the serial sections (red arrows in the right panel). St, stroma; T, tumor.

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Immunohistochemical analysis of VEGF-A and CD31 in lungs containing LLC colonies.  Finally, we examined VEGF-A and CD31 expression in tumors and endothelial cells by immunohistochemistry. Intense staining for VEGF-A was apparent in endothelial cells near the metastatic locus (Fig. 5c, upper left panel, blue arrows) together with stromal cells (Fig. 5c, upper left panel, white circle) in WT mice. By contrast, less-marked staining was found in EP3−/− mice even in the endothelial cells (Fig. 5c, upper right panel, blue arrows). Marked angiogenesis was observable mainly at the edge of metastatic foci in WT mice (Fig. 5c, lower left panel, red arrows), whereas in the CD31-positive vessels hardly any angiogenesis was detected in EP3−/− mice (Fig. 5c, lower right panel). These results indicated that VEGF-A expression not only in the endothelial cells but also in the stromal cells is EP3-dependent, and that angiogenesis in the stroma around tumors is also regulated by EP3 signaling. In addition, CD31-positive vessels were also stained for MMP-9 in WT mice (Fig. 5d). Thus, EP3-dependent development of the metastatic locus may be explained by enhanced neovascularization together with the upregulation of MMP-9 (Fig. 6).

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Figure 6.  Roles of EP3 signaling in enhancement of MMP-9 and VEGF-A expression in lungs containing Lewis lung carcinoma colonies in mice. The expression of MMP-9 in the endothelial cells but not in tumor stromal cells around the metastatic lesions was dependent on EP3 signaling, whereas that of VEGF-A both in the endothelial cells and in the stromal cells was dependent on EP3 signaling. The difference in metastatic colony formation between EP3−/− and EP3+/+ may be explained by EP3 dependency in these factors. PGE2, prostaglandin E2.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

MMP have long been associated with cancer-cell invasion and metastasis in the host.(19–21) Basic cancer research has focused mainly on mutations in cancer cells that result in either gain of function in oncogenes or loss of function in tumor-suppressor genes. However, the ECM of tumors and the non-cancerous stromal cells around the tumors also have an important impact on tumor progression. MMP can regulate the tumor microenvironment, and their expression and activation is increased in many human cancers compared with normal tissue. The MMP family is a group of proteolytic enzymes, and the major component of the basement membrane, type IV collagen, serves as a substrate for MMP-2 and MMP-9 (72- and 92-kDa type IV collagenases, respectively). The potencies in neoplastic epithelial invasion and metastasis have been reported to be correlated with the overexpression of these MMP.(12) In the present study, we focused on MMP expression because there have been reports describing an increase in MMP-2 and MMP-9 expression and metastatic potential in COX-2-overexpressing colon cancer cells and the involvement of MMP in metastasis of non-small cell lung carcinoma.(4,22,23) The expression of COX-2 and PGE2 synthesis on cell migration, the secretion of MMP, and the adhesion of human hepatoma cell lines have been investigated by others. A close correlation was observed between the expression of COX-2 under basal conditions and the secretion of MMP-2 and MMP-9. When primary cultures of fetal hepatocytes were treated with proinflammatory cytokines, lipopolysaccharide, and hepatocyte growth factor, the expression of COX-2 and the synthesis of high amounts of prostaglandins were increased with concomitant increases in the release of the active forms of MMP-2 and MMP-9 to the extracellular medium. This process was inhibited when the synthesis of prostaglandins was suppressed pharmacologically with COX-2 inhibitors. The same was true in prostate cancer.(24) We have analyzed LLC colony formation and the pro-MMP-9 level in the lungs on COX-2 inhibitor-treated mice. Mice treated with COX-2 inhibitors significantly inhibited LLC colony formation and pro-MMP-9 level. This suggested that, in our model, COX-2 inhibitors are conductive to tumor metastasis, which is associated with the level of MMP-9.

We have shown that COX-2-dependent prostaglandin formation is closely related to the facilitation of tumor metastasis. This was mainly through the signaling of EP3, a receptor of PGE2, as EP3−/− mice showed reduced colony formation with the same magnitude of inhibition as the COX-2 inhibitor and a nonselective COX inhibitor. In spite of the injection of a fixed number of LLC cells, the number of lung colonies formed in EP3−/− mice was significantly fewer than that in WT mice. These findings clearly indicated that the host EP3 receptor was relevant to the facilitation of metastasis in the lungs. EP3 signaling in the endothelial cells where the injected LLC cells were growing may be responsible for colony formation, as immunohistochemical studies have revealed that upregulation of MMP-9 in the host endothelial cells, but not in interstitial cells around the metastatic tumors, may be observed during colony formation. The host EP3 receptors, which were absent in EP3−/− mice, are key regulators of upregulation of MMP-9 and metastasis.

Furthermore, we have shown here that the enhanced angiogenesis in the lungs containing LLC colonies was EP3-dependent. This may be due to the upregulation of VEGF-A in the metastatic sites as we determined in this study. VEGF-A has been reported to be a major regulator of metastasis via upregulation of angiogenesis. In the present study, we showed that EP3 signaling clearly increased the angiogenesis seen around the metastatic colonies. This EP3 action on metastasis-associated angiogenesis was consistent with the roles of EP3 signaling in the syngeneic tumor implantation models. VEGF-A-positive cells in the stromal tissues around tumors were upregulated in both models. In fact, the reduction of VEGF-A in EP3−/− mice was confirmed in this lung metastasis model. This result certainly implies that VEGF-A plays significant roles in metastatic colony formation and/or development. It is quite interesting that the site of upregulation in response to EP3 signaling differed between MMP-9 and VEGF-A, as mentioned below. A recent study showed that MMP-9-expressing tumor-associated stromal cells such as macrophages play a key role in prepping premetastatic sites for eventual malignant cell growth in a manner dependent upon VEGF receptor (VEGFR)-1.(25) This suggested that VEGF-A generated around tumors or in the peripheral circulation, as shown in this study, may facilitate the recruitment of VEGFR-1-positive cells. Furthermore, we recently reported that EP3 signaling plays a significant role in the recruitment of VEGFR-1- and VEGFR-2-positive cells from bone marrow into the stromal tissues after implantation of LLC in mice.(26) These findings suggested that VEGF generated around tumors or in the peripheral circulation, as shown in this study, may facilitate the recruitment of VEGFR-1-positive cells. We have already reported that adenylate cyclase activation or increased cAMP levels were relevant to the increase in angiogenic response in a sponge implantion model, and that EP3 signaling activated AP-1, a transcription factor that is important for tumor metastasis and angiogenesis, in the cell fraction in the stroma, where angiogenesis was observed.(27) We had not anticipated this but several papers have reported that tumor-induced angiogenesis and metastasis are suppressed by inhibiting AP-1 activation, which leads to decreased regulation of MMP-9 and VEGF-A production.(28,29) It remains unclear, but there is the possibility that EP3−/− mice experience less tumor metastasis and angiogenesis due to suppressed AP-1 activation. As we have also stated previously, one of the splicing variants of EP3 receptors may be linked to adenylate cyclase activation, although EP2 and EP4 have been reported to activate this enzyme. Others have reported that inhibitors of protein kinase A, p38 MAP kinase, phosphatidylinositol-3-kinase, and nuclear factor-κB activation impair the release of MMP in response to PGE2 challenge, indicating the involvement of multiple steps in this process.(29) In agreement with this data, we have previously observed accumulation of MMP-2 and MMP-9 in fetal hepatocytes treated with lipopolysaccharide and hepatocyte growth factor (HGF).(30) In addition to fetal hepatocytes, the regulation of the expression of MMP has been described through a PGE2–cAMP-dependent pathway in monocytes, sinovial fibroblasts, and vascular endothelial cells.(31–33) We detected that EP3 signaling induced tumor metastasis and angiogenesis by expression of MMP-9 and VEGF-A. Interestingly, CD31-positive vessels were also stained for MMP-9 in WT mice. This indicated that the intracellular signaling used in the endothelial cells to upregulate MMP-9 expression and in the stromal cells to increase VEGF-A expression did not differ from each other.

In conclusion, host endothelial cell PGE2–EP3 signaling appears to be critical for tumor metastasis and angiogenesis. The effect of COX inhibitors suggested that COX-2 is the enzyme responsible for PGE2 formation. EP3 signaling on the endothelial cells was relevant to the induction of the proteinase and potent proangiogenic growth factor MMP-9 and VEGF in endothelial cells. Upregulated MMP-9 and VEGF certainly have invasive, metastatic, and proangiogenic actions. A highly selective EP3 antagonist exhibits chemopreventive action on endothelial cells and may become a novel therapeutic tool for cancer. EP3 antagonists may be useful to prevent the recurrence of lung cancer.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We would like to thank Michiko Ogino, Hanamo, Keiko Nakamigawa, and Osamu Katsumata for their technical assistance. This work was supported by a grant from the Integrative Research Program of the Graduate School of Medical Science, Kitasato University School of Medicine, and by a Grant from the Parent’s Association of Kitasato University School of Medicine. It was supported also by #12470529 and #12670094 High-tech Research Center grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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