Cancer/stroma interplay via cyclooxygenase-2 and indoleamine 2,3-dioxygenase promotes breast cancer progression

Human breast cancer cell lines MCF-7 and MDA-MB-231 were purchased from the Bioresource Collection and Research Center (BCRC) and ATCC. Immortalized human breast fibroblasts, RMF-EG [16], were kindly provided by Dr. Charlotte Kuperwasser (Tufts University, Boston, MA, USA). These cells were cultured in DMEM/F12 containing 10% fetal bovine serum (FBS). Other experimental materials and procedures are provided in Additional file 1.

To establish an inducible COX-2-expression cell line, MCF-7 cells (1 × 10⁶) were resuspended in buffer R containing 2 μg pCMV-Tet3G plasmid. Transfection was performed by using Neon microporation transfection system at room temperature with 1,250 V, 20 milliseconds, and two pulses. After 48 hours, the cells were selected with 1 mg/ml G418 for 2 weeks.

For the delivery of the second plasmid, pCMV-Tet3G stably transfected cells (1 × 10⁶) were resuspended in buffer R containing 2 μg of pTRE-mCherry-COX-2 plasmid. Transfection was performed by using Neon microporation transfection system at room temperature with 1,250 V, 20 milliseconds, and two pulses. After 48 hours, the cells were subjected to selection with 100 μg/ml hygromycin B. The stable cell line harbors both pCMV-Tet3G and pTRE-mCherry-COX-2 plasmid was used for induction experiment. The cells were maintained at 37°C in a 5% CO₂-humidified atmosphere and were incubated with doxycycline to induce COX-2 expression before co-culture assay.

In the co-culture system, 1 × 10⁵ RMF-EG cells were grown in the bottom of a six-well plate in DMEM/F12 with 10% FBS, and 1 × 10⁶ breast cancer cells were seeded on the 0.4-μm polyester membrane of a transwell insert in the same medium. MCF-7 cells were treated with or without doxycycline (1 μg/ml) for 72 hours. The conditioned medium, breast cancer cells, and RMF-EG cells were harvested for metabolomics and Western blotting analysis.

The proteins in the conditioned medium were removed by using 3-kDa ultracentrifugation filter devices. The metabolites in the filtered medium were extracted by using iced 50% methanol and were subsequently dried by a speedvac. Metabolite profiles were analyzed with the Metabolomics Core of National Health Research Institutes by using a high-resolution ultraperformance liquid chromatography (UPLC) coupled online to a triple-quadrupole time-of-flight mass spectrometry system, as described previously [17]. Metabolite identity was predicted with Human Metabolome Database [18].

Total RNA was isolated from cells by using an RNA extraction kit (Qiagen, Valencia, CA, USA) and 1 μg of RNA was reverse-transcribed to cDNA. Target mRNAs were quantified by using real-time PCR reactions with SYBR green fluorescein, and actin served as an internal control. cDNA synthesis was performed at 95°C for 3 minutes, and the conditions for PCR were 28 cycles of denaturation (95°C/1 minute), annealing (60°C/1 minute) extension (72°C/1 minute), and 1 cycle of final extension (72°C/10 minutes). The primers used are tryptophan 2,3-dioxygenase (TDO)-forward: 5′-GGGAACTACCTGCATTTGGA-3′; TDO-reverse: 5′-GTGCATCCGAGAAACAACCT-3′; IDO-forward: 5′-GCGCTGTTGGAAATAGCTTC-3′; IDO-reverse: 5′-CAGGACGTCAAAGCACTGAA-3′; E-cadherin-forward: 5′-CCTGGGACTCCACCTACAGA-3′; E-cadherin-reverse: 5′-GGATGAACACAGCGTGAGAGA-3′; actin-forward: 5′-TGTTACCAACTGGGACGACA-3′; actin-reverse: 5′-GGGGTGTTGAAGGTCTCAAA-3′.

MCF-7or COX-2-overexpressing MCF-7 cells were treated with or without 100 μM kynurenine for 24 hours; the cells were harvested with an RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM EDTA, and 50 mM NaF), and cellular lysates were incubated with anti-AhR antibody overnight at 4°C with rotation. Immunocomplexes were pulled down by Protein-G agarose bead, washed with RIPA buffer 3 times, and eluted with a sample buffer in boiled water for 10 minutes. The eluted samples were subjected to SDS-PAGE separation, and proteins were transferred to nitrocellulose membranes. Finally, the blots were probed with anti-E-cadherin or anti-Skp2 antibody and developed with enhanced chemiluminescence reagent.

Migration assays were conducted in transwells with 8-μm-pore filter inserts. Then 1 × 10⁴ MCF-7 or COX-2-overexpressing MCF-7 cells were seeded in the upper chamber. The lower chambers were filled with DMEM medium containing 1% FBS and 100 μM kynurenine. After 24 hours, the cells on the upper surface were removed by wiping with a cotton swab, and the cells that migrated to the lower surface were fixed. The cells were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the cell number in 15 randomly selected fields was counted under a microscope (100×). Experiments were performed independently at least 3 times.

MCF-7 cells treated with or without kynurenine were incubated with the proteasome inhibitor MG132 or the lysosome inhibitor chloroquine. The cells were harvested with a lysis buffer (20 mM Tris–HCl at pH 7.5, 150 mM sodium chloride, 1 mM calcium chloride, and 1% Triton X-100 and protease inhibitors), and cellular lysates were incubated with an E-cadherin antibody overnight at 4°C with rotation. Protein-G beads were added to the samples and incubated for another 1 hour at 4°C. Immunocomplexes were eluted and were subjected to SDS-PAGE separation, and proteins were transferred to nitrocellulose membranes. Finally, the blots were probed by using an anti-ubiquitin antibody to detect the ubiquitination status of E-cadherin.

MCF-7 or MCF-7-COX2 (8 × 10⁶) cells were mixed with RMF-EG (6 × 10⁶) cells in Hanks balanced salt solution and Matrigel (BD Transduction Laboratories, San Jose, CA, USA). Cells were inoculated into the fourth mammary fat pads of 6-week-old female BALB/cAnN.Cg-Foxn1nu/CrlNarl mice. Before the inoculation of the cancer cell/fibroblast mixture, all mice were primed with 6 mg/kg of 17β-estradiol twice a week for 3 weeks.

After inoculation, 17β-estradiol was continuously given to mice throughout the experiments. Measurement of tumor growth was begun at 4 weeks after injection, and tumor volume was calculated by using the equation: tumor volume = (length × width²)/2. After 10 weeks, mice injected with COX-2-overexpressing MCF-7 and RMF-EG produced tumors with volumes approximately 200 mm³ and were randomly divided into four groups that received vehicle (DMSO), NS-398 (10 mg/kg), L-1-methy-tryptophan (10 mg/kg), or both inhibitors 5 times per week.

Two weeks later, animals were killed, and tumors were isolated from mice. The statistical difference between experimental groups was evaluated with repeated-measures two-way ANOVA analysis. The animal-use protocol was approved by the Institutional Animal Care and Use Committee of National Health Research Institutes.

Paraffin-embedded human breast tumor tissues were obtained from Chi-Mei Medical Center (Tainan, Taiwan) between 1998 and 2004. The slides were stained with anti-COX-2 or anti-IDO antibodies. The COX-2 and IDO stainings were interpreted by using the H-score, defined by the following equation: H-score = ΣPi (i + 1), as previously described [19], where i is the intensity of the stained tumor cells (0 to 3+), and Pi is the percentage of stained tumor cells with various intensities. We classified tumors with cancer cells and stromal cells showing H-scores no less than the median of all scored cases as having high COX-2 and IDO expression, respectively.

The follow-up duration ranged from 5.4 to 143.6 months, with a mean of 87.1 months. Survival analyses for disease-specific and metastasis-free survival were performed by using Kaplan-Meier plots and compared by using the log-rank test. The correlation between COX-2 and IDO expression with clinicopathologic parameters was examined with χ² test. P value < 0.05 was considered statistically significant. This study was approved by the Research Ethics Committee of National Health Research Institutes. Written informed consent was obtained from all patients participating in this study.

We analyzed the metabolite profile of the supernatant of RMF-EG human breast fibroblasts co-cultured with MCF-76 or COX-2-overexpressing MCF-7 cells and found that several metabolites were increased in the supernatant of COX-2-overexpressing MCF-7/RMF-EG co-culture. A peak with the m/z ratio of 209 was increased about twofold (Figure 1A). By using Human Metabolome Database search, a candidate metabolite was predicted to be kynurenine. UPLC/MS/MS analysis demonstrated that fragmentation of kynurenine standard yielded three peaks with m/z ratio of 209, 192, and 146, which is consistent with the reported data (accession: K0009019, MassBank, [20]) (Figure 1B). Significant increase of kynurenine was confirmed with an ELISA assay (Figure 1C).

The rate-limiting enzymes in the generation of kynurenine are indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). We found a 2.5-fold of increase of IDO mRNA in RMF-EG cells co-cultured with COX-2-overexpressing MCF-7 cells, whereas the expression of TDO was not changed (Figure 1Di). A similar increase of IDO protein level was also found (Figure 1Dii). IDO was very low or undetectable in MCF-7- and COX-2-overexpressing MCF-7 cells, indicating that the kynurenine in the co-cultured medium was produced mainly by RMF-EG cells (Figure 1E). Co-culture of the COX-2-overexpressing MDA-MB-231 cells also upregulated IDO expression in RMF-EG cells (Figure 1F). These data suggest that COX-2-overexpressing breast cancer cells stimulate IDO expression and increase kynurenine secretion in co-cultured fibroblasts.

We found that PGE₂ increased IDO mRNA and protein levels in RMF-EG cells (Figure 2Ai and 2Aii). In addition, our data showed that only PGE₂-alcohol (an EP4 agonist) significantly upregulated IDO expression (Figure 2B). Knockdown of EP4 abolished PGE₂-induced increase of IDO in these cells (Figure 2Ci and 2Cii). By using different IDO deletion promoters, we demonstrated that PGE₂ stimulated IDO transcription via the −1,140/-844 region from the transcription start site (see Additional file 2: Figure S1). This region contained two potential γ-interferon-activated sites (GASs) that could be activated by different signal transducer and activator of transcription (STAT) proteins [21],[22]. Both STAT1 and STAT3 have been implicated in the regulation of IDO expression [23],[24].

We performed a ChIP assay and found that the binding of STAT3 to IDO promoter was increased in RMF-EG cells co-cultured with COX-2-overexpressing MCF-7 cells, whereas the binding of STAT1 was decreased (see Additional file 3: Figure S2). Knockdown of STAT3 abolished the increase of IDO induced by the EP4 agonist in RMF-EG cells (Figure 2D). Ectopic expression of STAT3 upregulated IDO (2.8-fold) in these cells (Figure 2E). Thus, COX-2-overexpressing breast cancer cells upregulate IDO expression in fibroblasts through the PGE₂/EP4/STAT3 pathway.

We next studied the effect of kynurenine on breast cancer cells. Kynurenine did not affect the proliferation of MCF-7 cells (Figure 3A). However, kynurenine significantly increased the motility of MCF-7 and COX-2-overexpressing MCF-7 cells (Figure 3B). The conditioned medium of RMF-EG fibroblasts preincubated with COX-2-overexpressing MCF-7 cells also increased the motility of MCF-7 cells (Figure 3C). We used 1-methyl-L-tryptophan to inhibit IDO activity in RMF-EG fibroblasts induced by co-culture with COX2-overexpressing MCF-7 cells and found that the stimulatory effect on cell motility was blocked (Figure 3C). These data suggested that kynurenine released by IDO-expressing fibroblasts enhanced the migration of breast cancer cells.

We investigated the expression of epithelial-to-mesenchymal markers in kynurenine-treated breast cancer cells and found that E-cadherin was reduced in a time-dependent manner (Figure 3Di). E-cadherin began to decrease around 8 hours after addition of kynurenine, and a 70% of reduction was found at 24 hours. However, its mRNA did not decrease substantially (Figure 3Dii). We found that kynurenine induced degradation of E-cadherin protein via a proteasome-dependent pathway, which could be rescued by MG132 (proteasome inhibitor) but not chloroquine (lysosome inhibitor) (Figure 3E). In addition, ubiquitination of E-cadherin protein was increased in kynurenine-treated MCF-7 cells (Figure 3F). These data suggest that kynurenine induces ubiquitination and degradation of E-cadherin to promote breast cancer cell motility.

Kynurenine has been shown to be an endogenous tumor-promoting ligand of the human AhR [25]. In addition, AhR is involved in the degradation of sex steroid receptors via a cullin 4B-dependent ubiquitination pathway [26]. We tested whether kynurenine reduced protein stability of E-cadherin through activation of AhR and found that the binding between AhR and E-cadherin was increased in kynurenine-treated MCF-7 cells (Figure 4A). Interestingly, Skp2, an F-box protein of the SCF E3 ligase, was co-immunoprecipitated with AhR, and the interaction was also increased by kynurenine. We did not detect the cullin 4B protein in the complex (data not shown). This is not a cell line-specific effect, because the interaction between AhR and E-cadherin protein was also elevated in kynurenine-treated A549 cells (see Additional file 4: Figure S3). The 3′-methylcholanthrene (3-MC), another AhR ligand, also induced co-localization of AhR and E-cadherin at the cell membrane (Figure 4B). Knockdown of Skp2 reversed kynurenine-induced reduction of E-cadherin protein without affecting AhR expression (Figure 4C). The AhR antagonist, 3′4′-dimethoxyflavone (3′4′-DMF), also inhibited the decrease of E-cadherin induced by kynurenine (Figure 4D). Additionally, kynurenine-increased migration of MCF-7 cells was blocked by 3′4′-DMF (Figure 4E). These data suggest that kynurenine induces the formation of E-cadherin/AhR/Skp2 complex and causes E-cadherin degradation.

The correlation between COX-2 expression in tumor tissues and IDO expression in CAFs was confirmed by two approaches. First, we isolated CAFs from two breast tumor tissues without or with COX-2 overexpression and found that IDO expression in CAFs was upregulated in COX-2-overexpressing tumor (see Additional file 5: Figure S4). Second, we used immunohistochemical analysis to detect COX-2 and IDO expression in a cohort of breast cancer tissues (Figure 5A). COX-2 expression in tumors was positively correlated with a high IDO expression in CAFs (65 of 101, 64%; P < 0.001) (Table 1). COX-2 was highly expressed in stage III (19 of 24, 79%; P < 0.05), N1-N2 (61 of 85, 71%; P < 0.001), and T3-4 stage (20 of24, 83%; P < 0.05) tumor specimens. IDO expression in CAFs was significantly expressed in stage III (20 of 24, 83%; P < 0.05), N1-N2 (60 of 85, 71%; P < 0.001), and T3-4 stage (21 of 24, 88%; P < 0.001) tumor specimens. The disease-specific and metastasis-free survival declined significantly in patients with high COX-2 expression in breast tumors (P = 0.0043 and P < 0.0001, respectively) (Figure 5B and Table 2). Similarly, the disease-specific and metastasis-free survival declined significantly in patients with high IDO expression in CAFs (P = 0.0045 and P < 0.0001) (Figure 5C and Table 2). More important, high COX-2 in tumors and high IDO1 expression in CAFs predicted worse disease-free and metastasis-free survivals in breast cancer patients (P < 0.0001, Figure 5D).

The effect of COX-2 and IDO inhibitors was evaluated in an orthotopic model. Inoculation of MCF-7/RMF-EG- or COX-2-overexpressing MCF-7/RMF-EG cell mixture induced tumors in nude mice primed with 17β-estradiol injection (Figure 6A). Tumor growth was higher in the COX-2-overexpressing MCF-7/RMF-EG group, and a 2.4-fold of increase of tumor volume was detected at 10 weeks (P < 0.01). The COX-2-overexpressing MCF-7/RMF-EG group was randomly divided into four subgroups (n = 3). Intratumoral injection of vehicle (DMSO, control), 10 mg/kg of NS398, 10 mg/kg of 1-methyl-L-tryptophan, or both inhibitors was conducted, and treatment was continuous for another 2 weeks. As shown in Figure 6B, tumor volume of the groups treated with NS398 or 1-methyl-L-tryptophan was smaller than that of the control group. Co-treatment of COX-2 and IDO inhibitor induced a more obvious reduction in tumor size, although it did not show an additive effect.