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Originally published In Press as doi:10.1074/jbc.M710038200 on March 4, 2008

J. Biol. Chem., Vol. 283, Issue 17, 11155-11163, April 25, 2008
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Cyclooxygenase-2 Up-regulates CCR7 via EP2/EP4 Receptor Signaling Pathways to Enhance Lymphatic Invasion of Breast Cancer Cells*Formula

Mei-Ren Pan{ddagger}, Ming-Feng Hou§, Hui-Chiu Chang{ddagger}, and Wen-Chun Hung||**1

From the {ddagger}Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, the §Department of Surgery, Kaohsiung Medical University, Kaohsiung 807, ||Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung 804, and **Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan 701, Taiwan

Received for publication, December 10, 2007 , and in revised form, February 29, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies demonstrate that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis. However, the mechanism by which COX-2 increases the invasion of cancer cells to lymph node is unclear. CCR7 is a chemokine receptor that plays important roles in the mediation of migration of leukocytes and dendritic cells toward lymphatic endothelial cells (LECs) that express receptor ligand CCL21. We found that treatment of prostaglandin E2 or ectopic expression of COX-2 in MCF-7 cells up-regulated CCR7 expression. On the contrary, knockdown of COX-2 by small hairpin RNA reduced CCR7 in COX-2-overexpressing MDA-MB-231 cells. Interaction of CCR7 and CCL21 was important for the migration of breast cancer cells toward LECs because antibodies against these two molecules inhibited the migration. We also found that COX-2 increased CCR7 expression via the EP2 and EP4 receptor in breast cancer cells. EP2 and EP4 agonists stimulated CCR7 in MCF-7 cells, whereas antagonists or small hairpin RNA of EP2 and EP4 attenuated CCR7 in MDA-MB-231 cells. Protein kinase A and AKT kinase were involved in COX-2-induced CCR7. Pathological analysis demonstrated that COX-2 overexpression was associated with CCR7, EP2, and EP4 expressions in breast tumor tissues. In addition, CCR7 expression in COX-2-overexpressing tumors was significantly correlated with lymph node metastasis. Collectively, we suggest that CCR7 is a down-stream target for COX-2 to enhance the migration of breast cancer cells toward LECs and to promote lymphatic invasion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenases (COXs)2 are the rate-limiting enzymes that catalyze the conversion of arachidonic acid to prostaglandins (PGs). Two COX isoforms with distinct tissue distributions and physiological functions have been identified (1, 2). Cyclooxygenase-1 (COX-1) is constitutively expressed in many tissues and plays important roles in the control of homeostasis (3). Conversely, COX-2 is an inducible enzyme and is activated by extracellular stimuli such as growth factors and pro-inflammatory cytokines (4). Recent investigations indicated that overexpression of COX-2 is frequently found in many types of cancer, including colon, lung, breast, pancreas, and head and neck cancers (5–9), and is usually associated with poor prognosis and short survival (10–12).

The contribution of COX-2 to tumorigenesis has been intensively studied. Several mechanisms are considered to mediate the tumorigenic activity of COX-2. First, PGE2, the main metabolite of COX-2, is a growth promoter and may directly stimulate proliferation of cancer cells (13, 14). Second, COX-2 is an angiogenic stimulator and may increase the production of angiogenic factors and migration of endothelial cells (15, 16). Third, COX-2-derived PGE2 is an anti-apoptotic molecule that may prevent apoptosis induced by anti-cancer drugs (17–19). Fourth, PGE2 is an immunoregulatory molecule that may suppress the anti-tumor activity of natural killer cells and macrophages (20). Fifth, COX-2 expression may increase the invasive ability of tumor cells and is closely linked with lymph node metastasis (21, 22). Results of xenograft animal studies also indicated that inhibition of COX-2 decreased tumor growth and lymph node metastasis (23). Whereas the functional role of COX-2 in the promotion of tumor angiogenesis is well documented, the mechanism by which COX-2 enhances lymphangiogenesis and lymph node metastasis remains unknown. A possible mediator that participates in the COX-2-induced lymphangiogenesis is vascular endothelial growth factor-C (VEGF-C). Ectopic expression of COX-2 in breast and lung cancer cells up-regulates VEGF-C, which may stimulate VEGF receptor 3 (VEGFR3) on the surface of LECs to promote lymphangiogenesis (24, 25).

The interaction between chemokines and their cognate receptors is critical in tumor metastasis. Recent evidence demonstrates that the chemokine receptor CXCR4 and its ligand stroma-derived factor-1 are key regulators for the dissemination of cancer cells (26, 27). The chemokine receptor CCR7 is important for the adhesion and chemotaxis of leukocyte and dendritic cell toward lymph nodes. Since LECs express a high level of CCR7 ligands CCL19 and CCL21, up-regulation of CCR7 in cancer cells may promote the migration of cancer cells toward LECs and enhance lymph node invasion. In this study, we addressed the following objectives: (a) correlation of expression of COX-2 and CCR7 in breast cancer cell lines and tumor tissues, (b) whether COX-2 may up-regulate CCR7 expression in breast cancer cells and promote their migration toward LECs, and (c) the receptors and signaling pathways that mediate the induction of CCR7 by COX-2.


Figure 1
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  		FIGURE 1.
Regulation of CCR7 by PGE2 and COX-2. A, mRNA expression of COX-2 and CCR7 in MCF-7 and MDA-MB-231 (231) breast cancer cells was investigated by RT-PCR. The CCR7 protein level on the cell surface was examined by flow cytometry (lower panel). B, MCF-7 cells was stimulated with vehicle (V, 0.1% methanol) or 100 nmol/liter of PGE2 for 24 h, and RT-PCR was performed to investigate CCR7 mRNA expression. Flow cytometry was also carried out to examine the expression of CCR7 protein. C, MCF-7 cells were transfected with control (C) or COX-2 expression vector. After 48 h, COX-2 and CCR7 mRNA levels were determined by RT-PCR. CCR7 protein level was examined by flow cytometry (low panel). D, MDA-MB-231 cells were transfected with control (C) or COX-2 shRNA (sh) expression vector for 48 h. COX-2 and CCR7 mRNA levels were studied by RT-PCR, and the CCR7 protein level was also investigated (lower panel). E, MDA-MB-231 cells were treated with vehicle (V, 0.1% DMSO) or 20 µmol/liter of NS398 for 24 h, and CCR7 protein level was determined by flow cytometry.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Experimental Agents—MCF-7 and MDA-MB-231 breast cancer cells were obtained from the cell bank of the National Health Research Institute (Miaoli, Taiwan). Cells were cultured in Dulbecco's modified Eagle's medium/F-12 medium containing 10% charcoal-stripped fetal calf serum (FCS) and antibiotics. Lymphatic endothelial cells were provided by Dr. Hsei-Wei Wang (National Yang-Ming University, Taipei). PGE1 alcohol (catalog number 13020), butaprost (catalog number 13740), sulprostone (catalog number 14765), and AH6809 (catalog number 14050) were purchased from Cayman Chemical (Ann Arbor, MI). AH23848 (catalog number A8227), dibutyryl cAMP (catalog number D0627), U0126 (catalog number U012), and KT5720 (catalog number K3761) were purchased from Sigma. LY294002 (catalog number L9908) was purchased from A. G. Scientific, Inc. (San Diego). Antibody against human COX-2 (C-20) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CCR7 (MAB197) and anti-CCL21 (AF366) antibodies were purchased from R & D Systems (Minneapolis, MN). Antibodies against EP2 (catalog number 101750) and EP4 (catalog number 101775) receptor were obtained from Cayman Chemical (Ann Arbor, MI). AKT (catalog number 9272), phospho-T308-AKT (catalog number 9275) and phospho-S473-AKT (catalog number 9271) antibodies were obtained from Cell Signaling (Danvers, MA). Dominant-negative AKT expression vector was provided by Dr. Min-Liang Kuo (National Taiwan University, Taipei). The inducible expression vector MT-Rev, which expressed regulatory subunit of PKA, was provided by Dr. Stanley McKnight (University of Washington, Seattle). Antibody against the regulatory subunit of PKA was purchased from BD Biosciences.

Tumor Tissues—Seventy nine paired normal and breast tumor tissues were obtained from patients who underwent resection of tumors at the Department of Surgery, Chung-Ho Memorial Hospital, Kaohsiung Medical University. Detailed data about patient- and tumor-related variables were collected by reviewing the patients' medical charts. Before acquisition of these tissues, the investigational nature of this study was explained to patients, and informed consent was obtained. Portions of resected tissues were quickly placed into the RNAlater solution (Ambion, Austin, TX) and were subjected for isolation of genomic DNA, total RNA, and proteins by using TRIzol reagent according to the procedures of the manufacturer (Invitrogen).


Figure 2
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  		FIGURE 2.
Inhibition of migration of breast cancer cells toward LECs by anti-CCR7 and anti-CCL21 antibodies. A, MCF-7 cells were transfected with control (C) or COX-2 expression vector. After 48 h, cells were collected and subjected to in vitro invasion assay as described under "Experimental Procedures." Non-immune immunoglobulin (Ig) or anti-CCR7 antibody (5 µg/ml) was added with cells into the upper part of the transwell unit, and the conditioned medium of LECs was added in the lower part. Invaded cell number was determined at 24 h after cell seeding. *, p < 0.05 when anti-CCR7 group was compared with Ig group. B, MDA-MB-231 cells was incubated with Ig or anti-CCR7 antibody (5 µg/ml), and cell invasion was studied as described above. *, p < 0.05 when anti-CCR7 group was compared with Ig group. C, conditioned medium of LECs was pre-cleared with anti-CCL21 antibody (5 µg/ml) for 4 h at room temperature and then added into the lower part of the transwell unit. MCF-7 or MDA-MB-231 (231) cells were seeded on the upper part of the unit, and invaded cell number was counted at 24 h after cell seeding. *, p < 0.05 when anti-CCL21 group was compared with Ig group. D, MCF-7 and MDA-MB-231 stable transfectants expressing COX-2 or CCR7 shRNA were used for in vitro invasion assay. *, p < 0.05 when shCOX-2 or shCCR7 group was compared with control vector group. E, inhibition of CCR7 expression by shRNA in two breast cancer cell lines is shown.

 
Reverse Transcription (RT)-PCR and Quantitative RT-PCR—Total RNA was isolated and subjected to RT-PCR analysis as described previously (28). The primer sequences were CCR7 forward, 5'-GGACCTGGGGAAACCAAT-3', and CCR7 reverse, 5'-GCCAGGTTGAGCAGGTAGGT-3'; COX-2 forward, 5'-GTCCTTTCAAGGAGAATGGTGC-3', and COX-2 reverse, 5'-GGTCTGGTGCCTGGTCTGATGATG-3'; EP2 forward, 5'-CAACCTCATCCGCATGCAC-3', and EP2 reverse, 5'-CTCAAAGGTCAGCCTG-3'; EP4 forward, 5'-GGTCATCTTACTCATTGCCACC-3', and EP-4 reverse, 5'-AGATGAAGGAGCGAGAGTGG-3'; CCL21 forward, 5'-ACCCAAGGCAGTGATGGA-3', and CCL21 reverse, 5'-TTTGGAGCCCTTTCCTTTCT-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5'-GAGTCAACGGATTTGGTCGT-3', and GAPDH reverse, 5'-TGTGGTCATGAGTCCTTCCA-3'. Amplified cDNA products were run on 2% agarose gels, stained with ethidium bromide, and visualized under UV light. The signal intensities of COX-2, CCR7, EP2, and EP4 were measured by a densitometer and normalized to GAPDH signal intensity. In some experiments, the mRNA level of CCR7 was quantified by real time RT-PCR using iQ SYBR Green Supermix (Bio-Rad). GAPDH served as an internal control for total complementary DNA content. Samples were amplified by using the MJ Mini real time PCR detection system (Bio-Rad). The PCR primers for CCR7 were as follows: CCR7 forward, 5'-GGGGAAACCAATGAAAAGC-3', and CCR7 reverse, 5'-ACCTCATCTTGACACAGGCATA-3'.

Immunoblotting—Treated cells were washed with ice-cold phosphate-buffered saline and harvested in a lysis buffer as described previously (28). Equal amounts of cellular proteins were separated by SDS-PAGE on 10 or 12.5% gels. Proteins were transferred onto nitrocellulose membranes, and the blots were incubated with different primary antibodies. Enhanced chemiluminescence reagents were used to depict the protein bands on the membrane.


Figure 3
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  		FIGURE 3.
Mediation of COX-2-induced CCR7 expression by EP2 and EP4 receptor. A, total RNA was extracted from MCF-7 and MDA-MB-231 (231), cells and RT-PCR was performed to examine the expression of EP1–4 receptors. B, MCF-7 cells were stimulated with vehicle (V, 0.1% DMSO), sulprostone (Sul, 10 µmol/liter), butaprost (But, 10 µmol/liter), PGE1 alcohol (E1, 1 µmol/liter), and PGE2 (E2, 100 nmol/liter) for 24 h, and CCR7 expression was studied by quantitative RT-PCR. *, p < 0.05 when drug-treated group was compared with vehicle group. C, MDA-MB-231 cells were treated with vehicle (V, 0.1% DMSO), AH23848 (10 µmol/liter), or AH6809 (50µmol/liter) for 24 h, and quantitative RT-PCR was performed to investigate CCR7 mRNA expression. *, p < 0.05 when drug-treated group was compared with vehicle group. Flow cytometry was also carried out to study the CCR7 protein level (lower panel). D, MDA-MB-231 stable transfectants expressing EP2 shRNA (EP2i) or EP4 shRNA (EP4i). EP2 and EP4 protein level was studied by Western blot analysis (left upper panel). CCR7 mRNA level of control and EP2i and EP4i was investigated by quantitative RT-PCR (right upper panel). *, p < 0.05 when shRNA-treated group was compared with control vector group. Flow cytometry was also carried out to study the CCR7 protein level of control (con), EP2i, or EP4i cells (lower panel). E, invasive ability of control (con), EP2i, or EP4i cells was studied. *, p < 0.05 when shRNA-treated group was compared with control vector group.

 
Flow Cytometry—For detection of CCR7 protein expression on the cell surface, cells were washed with ice-cold phosphate-buffered saline and incubated with biotinylated anti-CCR7 antibody at 4 °C for 30 min. After washing, cells were incubated with fluorescein isothiocyanate-conjugated avidin and subjected to flow cytometric analysis as described previously (29).

shRNA Targeting—shRNAs carrying puromycin selection marker were purchased from the National RNAi Core Facility of Academic Sinica (Taiwan). The sequences used for targeting were as follows: COX-2, 5'-GCAGATGAAATACCAGTCTTT-3'; CCR7, 5'-CCCTTTCTTGTACGCCTTCAT-3'; EP4, 5'-ACCAGTTATATCAGCCAAGTT-3'; and EP2, 5'-GCCATGTATGAAGCCAAATAT-3'. Cells were transfected with the shRNA plasmids and selected by puromycin for 3 weeks. The expression of CCR7, EP2, EP4, and COX-2 was investigated, and the stable clones with the highest knockdown efficiency were used for study.

In Vitro Invasion Assay—In vitro invasion assay was performed by using 24-well transwell units with polycarbonate filters (pore size 8 µm) coated on the upper side with Matrigel (Discovery Labware) (30). MCF-7 and MDA-MB-231 cells were collected, and 3 x 103 cells in 100 µl of medium with control immunoglobulin or anti-CCR7 antibody (5 µg/ml) were placed in the upper part of the transwell unit and allowed to invade for 24 h. The lower part of the transwell unit was filled with LEC-conditioned medium. In some experiments, LEC-conditioned medium was first incubated with anti-CCR21 (5 µg/ml) to neutralize CCL21 and then placed on the lower part for invasion study. After incubation, non-invaded cells on the upper part of the membrane were removed with a cotton swab. Invaded cells on the bottom surface of the membrane were fixed in formaldehyde, stained with Giemsa solution, and counted under a microscope. Invasive ability of stable transfectants of MDA-MB-231 cells expressing COX-2, CCR7, EP2, or EP4 shRNA was also investigated.

Statistical Analysis—The associations between COX-2 and CCR7 with clinicopathological parameters were assessed using {chi}2 test and Fisher's exact test. The correlations between COX-2 expression with CCR7, EP2, and EP4 were examined by Spearman rank correlation. Paired t test was performed to test the association of COX-2 and CCR7 up-regulation in tumor tissues. Wilcoxon rank sum test was used for testing COX-2 and CCR7 levels of lymph node-negative tumors with lymph node-positive tumors. Statistical significance was defined as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Up-regulation of CCR7 by COX-2 in Breast Cancer Cell Lines—We first compared the expression of COX-2 and CCR7 in non-metastatic MCF-7 and highly metastatic MDA-MB-231 breast cancer cells. As demonstrated in Fig. 1A, RT-PCR analysis showed that CCR7 was highly expressed in COX-2-overexpressing MDA-MB-231 cells. Conversely, the expression of CCR7 was low in MCF-7 cells that expressed low levels of COX-2. To confirm the RT-PCR results, we performed flow cytometry to investigate the CCR7 protein level on the cell surface. A positive correlation between CCR7 mRNA and protein expression was seen in these two breast cancer cell lines (Fig. 1A). We next used different approaches to clarify whether COX-2 could regulate CCR7 expression in breast cancer cells. Since MCF-7 cells expressed low level of COX-2 and CCR7, we treated these cells with PGE2 and found that PGE2 increased CCR7 mRNA and protein expression in MCF-7 cells (Fig. 1B). Ectopic expression of COX-2 also increased CCR7 expression in these cells (Fig. 1C). MDA-MB-231 cells expressed high levels of COX-2; we used shRNA to knock down COX-2 expression, and our data showed that inhibition of COX-2 by shRNA induced down-regulation of CCR7 (Fig. 1D). Consistent with this result, a COX-2-specific inhibitor NS398 also reduced the expression of CCR7 in MDA-MB-231 cells (Fig. 1E). By manipulating the expression of COX-2 in different breast cancer cell lines, we concluded that COX-2 and its metabolite PGE2 might stimulate CCR7 expression.

CCR7 Expression Enhanced the Migration of Breast Cancer Cells toward LECs—We next addressed whether CCR7 is required for the migration of breast cancer cells toward LECs. Primary cultured LECs used in these assays expressed high levels of CCL21 (data not shown). Conditioned medium of LECs was placed on the lower part of a transwell unit, and breast cancer cells were added to the upper part in the absence or presence of anti-CCR7 antibody. As shown in Fig. 2A, the migration ability of MCF-7 was poor. Ectopic expression of COX-2 significantly enhanced the migration ability of MCF-7 cells that could be specifically blocked by the anti-CCR7 antibody. MDA-MB-231 cells were highly invasive, and the migration ability was also significantly attenuated by the anti-CCR7 antibody (Fig. 2B). Similar results were observed when a co-culture system was used by seeding the LECs on the lower part of the transwell unit (data not shown). This effect was not non-specifically caused by anti-CCR7 antibody because this antibody did not exhibit cytotoxic effect on breast cancer cells (supplemental Fig. 1). To verify the migration of breast cancer cells was induced by the interaction between CCR7 and CCL21, conditioned medium was pre-cleared by anti-CCL21 antibody and then used for the migration assay. As shown in Fig. 2C, we found that migration of MCF-7 and MDA-MB-231 cells into a lower unit was significantly reduced. We also used shRNA to knock down COX-2 or CCR7 expression in these two cell lines and found that migration ability of the stable transfectants was attenuated (Fig. 2, D and E). In addition, the reduction was more significant in MDA-MB-231 cells (Fig. 2D). Our data suggested that up-regulation of CCR7 by COX-2 in breast cancer cells enhanced their migration toward LECs.

PGE2 and COX-2 Up-regulated CCR7 via EP2 and EP4 Receptor—Four types of G protein-coupled EP receptor have been identified in mammalian cells. Therefore, we addressed which type of receptor was involved in PGE2-induced increase of CCR7. RT-PCR analysis demonstrated that MCF-7 and MDA-MB-231 expressed all types of EP receptor (Fig. 3A). Both cell lines expressed similar levels of EP1 and EP3. Conversely, EP2 and EP4 were up-regulated in MDA-MB-231 cells. Since MCF-7 cells expressed very low levels of endogenous COX-2, we used ligand stimulation assay to study the specific receptors that mediated the induction of CCR7 by COX-2. Addition of PGE2, butaprost (EP2 agonist), or PGE1 alcohol (EP4 agonist) up-regulated CCR7 expression by 2–3-fold in MCF-7 cells (Fig. 3B). Conversely, the EP1/EP3 agonist sulprostone had little effect. We next performed antagonist inhibition assay to test whether EP2 and EP4 receptors were involved in the autocrine induction of CCR7 in COX-2-overexpressing MDA-MB-231 cells. Our data demonstrated that the EP2 antagonist AH6809 and the EP4 antagonist AH23848 suppressed CCR7 expression in MDA-MB-231 cells (Fig. 3C). To clarify the role of EP2 and EP4 more clearly, we used shRNA to knock down EP2 and EP4 expression. As shown in Fig. 3D, knockdown of EP2 and EP4 protein led to down-regulation of CCR7 mRNA and protein level in MDA-MB-231. In addition, the invasive ability of EP2 or EP4 knockdown cells was significantly reduced (Fig. 3E). These results strongly suggested that COX-2 up-regulated CCR7 via EP2 and EP4 receptor in breast cancer cells.


Figure 4
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  		FIGURE 4.
Involvement of PKA in the induction of CCR7 by COX-2. A, MCF-7 cells were treated with vehicle (V, H2O) or dibutyryl cAMP (100 µmol/liter) for 4 h, and RT-PCR was performed to examine the expression of CCR7 mRNA. Cells were also incubated with dibutyryl cAMP for 24 h, and flow cytometry was performed to investigate the CCR7 protein level (lower panel). B, MCF-7 cells were pretreated without (–) or with KT5720 (10 µmol/liter) for 30 min and then stimulated with 100 µmol/liter of dibutyryl cAMP (+) for another 4 h. CCR7 expression was checked by RT-PCR. C, MDA-MB-231 cells were incubated with vehicle (V, 0.1% methanol) or KT5720 (10µmol/liter) for 24 h. CCR7 mRNA and protein levels were studied by RT-PCR and flow cytometry (lower panel). D, MDA-MB-231 were incubated with vehicle (V, 0.1% DMSO), U0126 (10 µmol/liter, U0), or LY294002 (10 µmol/liter, LY) for 24 h. RT-PCR was performed to investigate CCR7 expression.

 
PKA and AKT Were Involved in the Induction of CCR7 by COX-2—The common signaling effector activated by EP2 and EP4 receptor is PKA. Therefore, we investigated the functional role of PKA in COX-2-induced CCR7. Stimulation of MCF-7 cells by dibutyryl cAMP, a potent PKA activator, up-regulated CCR7 mRNA and protein expression (Fig. 4A). Forskolin, an adenylyl cyclase and PKA activator, also increased CCR7 expression in MCF-7 cells (supplemental Fig. 2). Pretreatment of a PKA inhibitor KT5720 potently suppressed dibutyryl cAMP-induced CCR7 (Fig. 4B). Similarly, KT5720 also attenuated CCR7 expression in MDA-MB-231 cells (Fig. 4C). To address the functional role of PKA more specifically, we ectopically expressed the regulatory subunit of PKA to inhibit its activity in MDA-MB-231 cells and found that it caused a decrease of CCR7 expression (supplemental Fig. 3). Moreover, the increase of PKA regulatory subunit in MCF-7 cells also reduced butaprost (EP2 agonist)-induced and PGE1 alcohol (EP4 agonist)-induced CCR7 expression (supplemental Fig. 4). EP4 receptor has been found to activate ERK via phosphatidylinositol 3-kinase (31). However, treatment of phosphatidylinositol 3-kinase and ERK inhibitor (LY294002 and U0126) did not affect CCR7 expression in MDA-MB-231 cells (Fig. 4D). A recent study demonstrated that stimulation of the EP2 receptor induced PKA-dependent and phosphatidylinositol 3-kinase-independent activation of AKT (31). In addition, PKA phosphorylated Thr-308 but not Ser-473 (two major activating phosphorylation sites) on AKT (32). Therefore, we studied whether AKT was involved in the induction of CCR7 by COX-2. Fig. 5A showed that PGE2 and dibutyryl cAMP increased the phosphorylation of Thr-308 of AKT in MCF-7 cells. Pretreatment of PKA inhibitor KT5720 totally abolished this effect. Our data also showed that PGE2-increased CCR7 expression in MCF-7 cells was attenuated by dominant-negative AKT (Fig. 5B). In addition, dominant-negative AKT also reduced CCR7 expression in MDA-MB-231 cells (Fig. 5C). PKA inhibitor KT5720 reduced Thr-308 but not Ser-473 phosphorylation of AKT in MDA-MB-231 cells (Fig. 5D). More importantly, EP2 and EP4 receptor antagonists AH23848 and AH6809 also inhibited Thr-308 phosphorylation. These data suggested that COX-2 induced CCR7 expression via a PKA/AKT-dependent pathway.

COX-2, CCR7, EP2, and EP4 Were Co-expressed in Breast Tumor Tissues—Our study on breast cancer cell lines clearly indicated that CCR7 was up-regulated by COX-2. In addition, we also found that EP2 and EP4 receptors were up-regulated in COX-2-overexpressing MDA-MB-231 cells. Therefore, we investigated whether COX-2 and CCR7 were co-expressed in breast tumor tissues as seen in breast cancer cell lines. Breast tumor tissues and their adjacent normal parts from 79 patients were studied. RT-PCR analysis of five tumor tissues is shown in Fig. 6. The expression levels of COX-2 and CCR7 of normal and tumor tissues were normalized with GAPDH and compared. We found that COX-2 and CCR7 were increased in 44.3% (35/79) and 51.9% (41/79) of breast tumor tissues, respectively. {chi}2 analysis indicated that CCR7 expression was significantly associated with COX-2 expression (p = 0.008; Table 1). COX-2 expression also significantly correlated with EP2 (p = 0.024) and EP4 (p = 0.008) expression. Spearman rank correlation test confirmed that COX-2 expression strongly associated with CCR7 (coefficient = 0.528, p = 0.000), EP2 (coefficient = 0.235, p = 0.037), and EP4 (coefficient = 0.229, p = 0.042). Our data indicated that COX-2 overexpression was associated with up-regulation of CCR7, EP2, and EP4 in breast cancer tissues as found in breast cancer cell lines.


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  		TABLE 1
Association of COX-2 and CCR7, EP2, and EP4 mRNA expression in breast tumor tissues

The signal intensities of COX-2, CCR7, EP2, and EP4 of normal or tumor tissues were normalized to GAPDH and compared. The associations between COX-2 and CCR7, and EP2 and EP4 were calculated by {chi}2 analysis, and the p value was shown. N, normal; U, up-regulated.

 


Figure 5
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  		FIGURE 5.
PKA-induced AKT activation involved in the induction of CCR7 by COX-2. A, MCF-7 cells were treated with vehicle (V, 0.1% DMSO), PGE2 (100 nmol/liter), or dibutyryl cAMP (100 µmol/liter) for 30 min, and the amount of T308-AKT and S473-AKT was assessed by phospho-specific antibodies, and the amount of AKT was used as internal control. Lower panel, cells were pretreated with KT5720 for 1 h and then stimulated with PGE2 or dibutyryl cAMP. The phosphorylation status of Thr-308 was studied. B, MCF-7 cells were transfected with control (C) or dominant-negative AKT (DN-AKT) expression vector for 48 h. Cells were then treated with vehicle (V) or PGE2 for another 24 h. CCR7 expression was assessed by flow cytometry. C, MDA-MB-231 cells were transfected with control (C) or dominant-negative AKT (DN-AKT) expression vector for 48 h, and CCR7 expression was determined by flow cytometry. D, MDA-MB-231 cells were treated with vehicle (V, 0.1% DMSO), KT5720 (10 µmol/liter), AH23848 (10 µmol/liter), or AH6809 (50 µmol/liter) for 24 h. The phosphorylation status of Thr-308 and Ser-473 was studied by phospho-specific antibodies, and the protein level of AKT was used as internal control.

 


Figure 6
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  		FIGURE 6.
Expression of COX-2, CCR7, EP2, and EP4 in paired normal and breast tumor tissues. Total RNA was isolated from breast tumor tissues and their normal parts and subjected to RT-PCR analysis to study the expression of COX-2, CCR7, EP2, and EP4.

 
COX-2 and CCR7 Were Up-regulated in Breast Tumor Tissues and Associated with Lymph Node Metastasis—Paired t test clearly indicated that expression of COX-2 and CCR7 was increased in tumor tissues (p = 0.031 and 0.047, respectively). So, we investigated the correlation of COX-2 and CCR7 with clinicopathological parameters. As shown in Table 2, COX-2 expression was not associated with tumor size, histological grade, progesterone receptor, HER-2/neu, and histological type. On the contrary, COX-2 expression was positively associated with lymph node metastasis (p = 0.030) and negatively correlated with estrogen receptor (p = 0.044). CCR7 expression was not correlated with tumor size, histological grade, HER-2/neu, and histological type. However, CCR7 was negatively correlated with estrogen receptor and progesterone receptor. The lack of association of CCR7 with lymph node metastasis was unexpected. We focused on the expression of COX-2 and CCR7 in tumor tissues and compared them in lymph node-negative and lymph node-positive tumors by using Wilcoxon rank sum test. Our data showed that COX-2 expression was more frequently found in tumors with lymph node metastasis (p = 0.040). Similar to the results of {chi}2 test, no significant association of CCR7 and lymph node metastasis was observed (p = 0.355). However, we found that CCR7 expression was significantly associated with lymph node metastasis (p = 0.048) in COX-2-overexpressing tumors when a serial testing was carried out. These data strongly suggested that CCR7 was a critical determinant to predict lymph node metastasis in COX-2-overexpressing tumors. Collectively, our results indicated that COX-2 and CCR7 were up-regulated in breast cancer and were associated with enhancement of lymph node metastasis.


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  		TABLE 2
Clinicopathological factors associated with COX-2 and CCR7 mRNA expression

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The importance of our study is that we provide a mechanistic insight by which COX-2 promotes breast cancer cells to invade into the lymphatic system. Previous studies have demonstrated that COX-2 expression was associated with lymph node metastasis in breast cancer (21, 22). In addition, COX-2 is a negative prognostic factor for disease-free survival and overall survival in patients with breast cancer (22). Therefore, it is predictable that COX-2 may contribute to enhancement of lymph node metastasis. However, the underlying mechanism is unknown. Recently, a candidate gene for the mediation of lymph node invasion or lymphangiogenesis by COX-2 has been suggested. Timoshenko et al. (25) showed that COX-2 expression was strongly correlated with VEGF-C expression in breast cancer. VEGF-C is one of the most potent growth factors for the induction of lymphangiogenesis because the LECs express high amounts of its cognate receptor VEGFR3. The authors showed that COX-2 acted via EP1 and EP4 receptor to increase VEGF-C expression. Furthermore, a selective COX-2 inhibitor celecoxib potently decreased tumor growth and recurrence by inhibiting COX-2-induced lymphangiogenesis (23). Similar results have also been found in human lung cancer cell lines and tumor tissues (24). Because COX-2-induced lymph node metastasis is a complex process, we think VEGF-C is not the only effector of COX-2. In this study, we identified another key player CCR7. A pioneer study demonstrated that the chemokine receptors were involved in breast cancer metastasis, and CCR7 was found to be one of the most consistent up-regulated chemokine receptors in breast tumor tissues (26). In agreement with these data, we found that CCR7 was up-regulated in 51.9% of breast tumor tissues. Another recent study showed that CCR7 was a novel biomarker for predicting lymph node metastasis in T1 breast cancer (33). Although the association of COX-2 and CCR7 with lymph node metastasis of breast cancer has been separately reported, the functional cross-link between these two genes in the mediation of lymph node metastasis was missing. We provide the first evidence that COX-2 acts via EP2 and EP4 receptor to up-regulate CCR7 expression in breast cancer cells. The increase of CCR7 was important for the migration and invasion of breast cancer cells toward LECs because anti-CCR7 antibody significantly attenuated this effect. Moreover, knockdown of CCR7 in COX-2-overexpressing MDA-MB-231 cells also resulted in reduction of LEC-induced migration (Fig. 2D). These data strongly suggest that CCR7 is a critical mediator for COX-2 to promote lymph node metastasis.

The signaling pathways that participated in COX-2-induced CCR7 is another important issue. We found that PKA and AKT were involved in the up-regulation of CCR7 by COX-2. Because activation of EP2 and EP4 increases intracellular cAMP, it is not surprising that PKA is a downstream effector for COX-2 to increase CCR7. Interestingly, similar observations had also been reported in dendritic cells (34). Dendritic cells are antigen-presenting cells and are able to migrate from peripheral tissues to lymph node (35). Scandella et al. (34) demonstrated that PGE2 increased cAMP level and CCR7 expression in dendritic cells. We extend these results by showing that AKT is a downstream mediator of PKA because PGE2, dibutyryl cAMP, and forskolin increased phosphorylation of Thr-308 of AKT, whereas PKA inhibitor KT5720 totally abolished this effect. In addition, ectopic expression of the regulatory subunit of PKA or dominant-negative AKT potently attenuated CCR7 expression. These results suggest that COX-2 acts via a PKA/AKT-dependent pathway to up-regulate CCR7. The most well defined transcription factor that controls CCR7 expression is NF-{kappa}B (36). The functional link between AKT and NF-{kappa}B is now under investigation in our laboratory.

An important finding of our pathological analysis is the association of COX-2 and CCR7 with lymph node metastasis. Our cell-based study indicates that CCR7 is one of the critical mediators for COX-2 to enhance lymph node invasion. However, the clinical relevance needs to be verified. We studied the expression of COX-2 and CCR7 in breast tumor tissues, and our results confirmed a strong correlation between COX-2 and lymph node metastasis. Since COX-2 exhibits proliferative, angiogenic, and anti-apoptotic activities, it is not surprising that COX-2 is associated with lymph node metastasis. However, we found that CCR7 alone is not significantly linked with lymph node metastasis. Conversely, CCR7 expression in COX-2-expressing tumors is an important predictor for lymph node metastasis. The results suggest that COX-2 may increase CCR7 expression and cooperate with CCR7 to promote lymph node metastasis. Interestingly, we also found that the CCR7 level was negatively associated with progesterone receptor and estrogen receptor. Whether steroid hormones like estrogen and progesterone may regulate CCR7 expression in dendritic cells or cancer cells is still unknown. It is a critical issue for future studies.

The results of this study are of great clinical significance. Several strategies for the prevention or treatment of lymph node metastasis in breast cancer are suggested. Because COX-2 is an upstream regulator for CCR7, targeting of COX-2 may possibly reduce CCR7 expression and attenuate metastasis. This hypothesis is strongly supported by our results (Fig. 1, D and E). We think non-steroidal anti-inflammatory drugs and COX-2 inhibitors are good candidates for the prevention or treatment of COX-2-induced lymph node metastasis because these drugs may simultaneously repress the expression of VEGF-C and CCR7 stimulated by COX-2. Another choice is the antibody or specific inhibitors that can block the interaction between CCR7 and its cognate ligand CCL21 and CCL19. Identification of the functional importance of stroma-derived factor-1/CXCR4 interaction in tumor metastasis has led to the intense development of drugs that may inhibit this interaction. Similar concepts can be adapted to the prevention of CCL21/CCR7 interaction. This hypothesis is supported by our results that anti-CCL21 and CCR7 antibodies potently suppress the migration of breast cancer cells toward LECs (Fig. 2, B and C). Collectively, we identify CCR7 as a downstream mediator for COX-2 to promote lymphatic invasion and suggest that inhibition of COX-2 and CCL21/CCR7 interaction may be helpful for the treatment of lymph node metastasis.


   FOOTNOTES
 
* This work was supported by the grants from the Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, and National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–4. Back

1 To whom correspondence should be addressed: 70, Lien-Hai Rd., Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan, Republic of China. Fax: 886-7-5250197; E-mail: hung1228{at}ms10.hinet.net.

2 The abbreviations used are: COX, cyclooxygenase; LECs, lymphatic endothelial cells; PG, prostaglandin; shRNA, small hairpin RNA; PKA, protein kinase A; VEGF-C, vascular growth factor C; VEGFR3, vascular growth factor receptor 3; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ERK, extracellular signal-regulated kinase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Smith, W. L., DeWitt, D. L., and Garavito, R. M. (2000) Annu. Rev. Biochem. 69, 145–182[CrossRef][Medline] [Order article via Infotrieve]
  2. Warner, T. D., and Mitchell, J. A. (2004) FASEB J. 18, 790–804[Abstract/Free Full Text]
  3. Morita, I. (2002) Prostaglandins Other Lipid Mediat. 68–69, 165–175
  4. Turini, M. E., and DuBois, R. N. (2002) Annu. Rev. Med. 53, 35–57[CrossRef][Medline] [Order article via Infotrieve]
  5. Sano, H., Kawahito, Y., Wilder, R. L., Hashiramoto, A., Mukai, S., Asai, K., Kimura, S., Kato, H., Kondo, M., and Hla, T. (1995) Cancer Res. 55, 3785–3789[Abstract/Free Full Text]
  6. Hida, T., Yatabe, Y., Achiwa, H., Muramatsu, H., Kozaki, K., Nakamura, S., Ogawa, M., Mitsudomi, T., Sugiura, T., and Takahashi, T. (1998) Cancer Res. 58, 3761–3764[Abstract/Free Full Text]
  7. Hwang, D., Scollard, D., Byrne, J., and Levine, E. (1998) J. Natl. Cancer Inst. 90, 455–460[Abstract/Free Full Text]
  8. Tucker, O. N., Dannenberg, A. J., Yang, E. K., Zhang, F., Teng, L., Daly, J. M., Soslow, R. A., Masferrer, J. L., Woerner, B. M., Koki, A. T., and Fahey, T. J. (1999) Cancer Res. 59, 987–990[Abstract/Free Full Text]
  9. Chan, G., Boyle, J. O., Yang, E. K., Zhang, F., Sacks, P. G., Shah, J. P., Edelstein, D., Soslow, R. A., Koki, A. T., Woerner, B. M., Masferrer, J. L., and Dannenberg, A. J. (1999) Cancer Res. 59, 991–994[Abstract/Free Full Text]
  10. Achiwa, H., Yatabe, Y., Hida, T., Kuroishi, T., Kozaki, K., Nakamura, S., Ogawa, M., Sugiura, T., Mitsudomi, T., and Takahashi, T. (1999) Clin. Cancer Res. 5, 1001–1005[Abstract/Free Full Text]
  11. Ristimaki, A., Sivula, A., Lundin, J., Lundin, M., Salminen, T., Haglund, C., Joensuu, H., and Isola, J. (2002) Cancer Res. 62, 632–635[Abstract/Free Full Text]
  12. Ohd, J. F., Nielsen, C. K., Campbell, J., Landberg, G., Lofberg, H., and Sjolander, A. (2003) Gastroenterology 124, 57–70[CrossRef][Medline] [Order article via Infotrieve]
  13. Pai, R., Soreghan, B., Szabo, I. L., Pavelka, M., Baatar, D., and Tarnawski, A. S. (2002) Nat. Med. 8, 289–293[CrossRef][Medline] [Order article via Infotrieve]
  14. Mutoh, M., Watanabe, K., Kitamura, T., Shoji, Y., Takahashi, M., Kawamori, T., Tani, K., Kobayashi, M., Maruyama, T., Kobayashi, K., Ohuchida, S., Sugimoto, Y., Narumiya, S., Sugimura, T., and Wakabayashi, K. (2002) Cancer Res. 62, 28–32[Abstract/Free Full Text]
  15. Tsujii, M., Kawano, S., Tsuji, S., Sawaoka, H., Hori, M., and DuBois, R. N. (1998) Cell 93, 705–716[CrossRef][Medline] [Order article via Infotrieve]
  16. Gately, S. (2000) Cancer Metastasis Rev. 19, 19–27[CrossRef][Medline] [Order article via Infotrieve]
  17. Lin, M. T., Lee, R. C., Yang, P. C., Ho, F. M., and Kuo, M. L. (2001) J. Biol. Chem. 276, 48997–49002[Abstract/Free Full Text]
  18. Sorokin, A. (2004) Curr. Pharm. Des. 10, 647–657[CrossRef][Medline] [Order article via Infotrieve]
  19. Chang, H. C., and Weng, C. F. (2001) Oncol. Rep. 8, 1321–1325[Medline] [Order article via Infotrieve]
  20. Huang, M., Sharma, S., Mao, J. T., and Dubinett, S. M. (1996) J. Immunol. 157, 5512–5520[Abstract]
  21. Costa, C., Soares, R., Reis-Filho, J. S., Leitao, D., Amendoeira, I., and Schmitt, F. C. (2002) J. Clin. Pathol. 55, 429–434[Abstract/Free Full Text]
  22. Denkert, C., Winzer, K. J., Muller, B. M., Weichert, W., Pest, S., Kobel, M., Kristiansen, G., Reles, A., Siegert, A., Guski, H., and Hauptmann, S. (2003) Cancer 97, 2978–2987[CrossRef][Medline] [Order article via Infotrieve]
  23. Barnes, N. L., Warnberg, F., Farnie, G., White, D., Jiang, W., Anderson, E., and Bundred, N. J. (2007) Br. J. Cancer 96, 575–582[CrossRef][Medline] [Order article via Infotrieve]
  24. Su, J. L., Shih, J. Y., Yen, M. L., Jeng, Y. M., Chang, C. C., Hsieh, C. Y., Wei, L. H., Yang, P. C., and Kuo, M. L. (2004) Cancer Res. 64, 554–564[Abstract/Free Full Text]
  25. Timoshenko, A. V., Chakraborty, C., Wagner, G. F., and Lala, P. K. (2006) Br. J. Cancer 94, 1154–1163[CrossRef][Medline] [Order article via Infotrieve]
  26. Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M. E., McClanahan, T., Murphy, E., Yuan, W., Wagner, S. N., Barrera, J. L., Mohar, A., Verastegui, E., and Zlotnik, A. (2001) Nature 410, 50–56[CrossRef][Medline] [Order article via Infotrieve]
  27. Taichman, R. S., Cooper, C., Keller, E. T., Pienta, K. J., Taichman, N. S., and McCauley, L. K. (2002) Cancer Res. 62, 1832–1837[Abstract/Free Full Text]
  28. Liu, L. T., Chang, H. C., Chiang, L. C., and Hung, W. C. (2003) Cancer Res. 63, 3069–3072[Abstract/Free Full Text]
  29. Berahovich, R. D., Lai, N. L., Wei, Z., Lanier, L. L., and Schall, T. J. (2006) J. Immunol. 177, 7833–7840[Abstract/Free Full Text]
  30. Chang, H. C., Cho, C. Y., and Hung, W. C. (2006) Cancer Res. 66, 8413–8420[Abstract/Free Full Text]
  31. Fujino, H., Xu, W., and Regan, J. W. (2003) J. Biol. Chem. 278, 12151–12156[Abstract/Free Full Text]
  32. Filippa, N., Sable, C. L., Filloux, C., Hemmings, B., and van Obberghen, E. (1999) Mol. Cell. Biol. 19, 4989–5000[Abstract/Free Full Text]
  33. Cabioglu, N., Yazici, M. S., Arun, B., Broglio, K. R., Hortobagyi, G. N., Price, J. E., and Sahin, A. (2005) Clin. Cancer Res. 11, 5686–5693[Abstract/Free Full Text]
  34. Scandella, E., Men, Y., Gillessen, S., Forster, R., and Groettrup, M. (2002) Blood 100, 1354–1361[Abstract/Free Full Text]
  35. Saeki, H., Moore, A. M., Brown, M. J., and Hwang, S. T. (1999) J. Immunol. 162, 2472–2475[Abstract/Free Full Text]
  36. Hegde, S., Pahne, J., and Smola-Hess, S. (2004) FASEB J. 18, 1439–1441[Abstract/Free Full Text]

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