Cancer/stroma interplay via cyclooxygenase-2 and indoleamine 2,3-dioxygenase promotes breast cancer progression
Breast Cancer Research
Cancer/stroma interplay via cyclooxygenase-2 and indoleamine 2,3-dioxygenase promotes breast cancer progression
Jing-Yi Chen 0
Chien-Feng Li 3
Cheng-Chin Kuo 2
Kelvin K Tsai 0
Ming-Feng Hou 1 4
Wen-Chun Hung 0 1
0 National Institute of Cancer Research, National Health Research Institutes , No. 367, Shengli Road, Tainan 704 , Taiwan
1 Cancer Center, Kaohsiung Medical University Hospital , Kaohsiung 807 , Taiwan
2 Institute of Cellular and System Medicine, National Health Research Institutes , Maoli 350 , Taiwan
3 Department of Pathology, Chi-Mei Foundation Medical Center , Tainan 710 , Taiwan
4 Department of Surgery, College of Medicine, Kaohsiung Medical University
Introduction: Expression of indoleamine 2,3-dioxygenase (IDO) in primary breast cancer increases tumor growth and metastasis. However, the clinical significance of stromal IDO and the regulation of stromal IDO are unclear. Methods: Metabolomics and enzyme-linked immunosorbent assay (ELISA) were used to study the effect of cyclooxygenase-2 (COX-2)-overexpressing breast cancer cells on IDO expression in co-cultured human breast fibroblasts. Biochemical inhibitors and short-hairpin RNA (shRNA) were used to clarify how prostaglandin E2 (PGE2) upregulates IDO expression. Associations of stromal IDO with clinicopathologic parameters were tested in tumor specimens. An orthotopic animal model was used to examine the effect of COX-2 and IDO inhibitors on tumor growth. Results: Kynurenine, the metabolite generated by IDO, increases in the supernatant of fibroblasts co-cultured with COX-2-overexpressing breast cancer cells. PGE2 released by cancer cells upregulates IDO expression in fibroblasts through an EP4/signal transducer and activator of transcription 3 (STAT3)-dependent pathway. Conversely, fibroblast-secreted kynurenine promotes the formation of the E-cadherin/Aryl hydrocarbon receptor (AhR)/S-phase kinase-associated protein 2 (Skp2) complex, resulting in degradation of E-cadherin to increase breast cancer invasiveness. The enhancement of motility of breast cancer cells induced by co-culture with fibroblasts is suppressed by the IDO inhibitor 1-methyl-tryptophan. Pathological analysis demonstrates that upregulation of stromal IDO is a poor prognosis factor and is associated with of COX-2 overexpression. Co-expression of cancer COX-2 and stromal IDO predicts a worse disease-free and metastasis-free survival. Finally, COX-2 and IDO inhibitors inhibit tumor growth in vivo. Conclusion: Integration of metabolomics and molecular and pathological approaches reveals the interplay between cancer and stroma via COX-2, and IDO promotes tumor progression and predicts poor patient survival.
Chronic inflammation is strongly associated with the
development of cancer [
]. One of the crucial mediators of
inflammatory reaction is cyclooxygenase (COX). The
COX family of enzymes comprises two members (COX-1
and COX-2) and is the main controller of eicosanoid
biosynthesis. Studies of human breast tumor tissues
demonstrate that upregulation of COX-2 has been detected in
approximately 40% of human breast tumor tissues, as well
as preinvasive ductal carcinoma in situ lesions [
Elevated expression of COX-2 is associated with large tumor
size, advanced histologic grade, axillary node metastasis,
and unfavorable disease-free survival [
]. In addition,
COX-2 expression also links with increased tumor
angiogenesis . Epidemiologic investigations suggest that
use of nonsteroidal antiinflammatory drugs or selective
COX-2 inhibitors reduces breast cancer risk [
Results of animal study also support an oncogenic role
of COX-2. Transgenic COX-2 overexpression induces
mammary tumor formation in mice [
tumorigenic transformation is highly dependent on PGE2
production and angiogenic switch. In addition, HER-2/Neu
oncogene-induced mammary tumorigenesis and
angiogenesis are dramatically attenuated in COX-2 knockout
mice, suggesting a key role of COX-2 in breast cancer
]. Recent studies also show that COX-2 inhibitors
exhibit antitumor and antiangiogenic activities in vivo
and exhibit chemopreventive effects against mammary
carcinogenesis induced by 7,12-dimethyl-benz(a)anthracene
in rats [
]. All of the results suggest that COX-2 is
involved in multiple steps of breast carcinogenesis and is
a potential target for cancer prevention and therapy.
Interplay between breast cancer cells and
cancerassociated fibroblasts (CAFs), the most abundant and
active stromal cells, is crucial for tumor growth,
progression, angiogenesis, and therapeutic resistance [
Cancer cells release a number of factors to enhance the
production of cytokines, chemokines, and matrix
metalloproteinases (MMPs) from CAFs, which in turn facilitate
cancer cell proliferation, migration, and invasion. Previous
study demonstrated that stromal fibroblasts present in
invasive breast carcinomas can secrete large amounts of
stromal cell-derived factor 1 (SDF-1) to enhance tumor
growth and angiogenesis [
]. Co-injection of breast
cancer cells and fibroblasts also promotes the progression of
ductal carcinoma in situ to invasive breast carcinoma by
stimulating chemokine (C-X-C motif ) ligand 14 (CXCL14)
and chemokine (C-X-C motif ) ligand 12 (CXCL12)
]. However, most studies addressing the
crosstalk between cancer and stromal cells focus on
protein factors like cytokines and chemokines. Whether
other small molecules such as lipids or metabolites
participate in cancer-stromal cell interaction is largely
The tumor-promoting role of CAFs via upregulation of
COX-2 in ductal carcinoma in situ of the breast was first
demonstrated by Hu et al. [
]. The authors showed that
co-culture with fibroblasts increases COX-2 expression in
breast cancer cells and subsequently induces MMP-9 and
MMP-14 in these cells to promote invasion. They also
elucidated the underlying mechanism by demonstrating
that inhibition of nuclear factor kappa-light-chain-enhancer
of activated B cells (NF-κB) and COX-2 activity reduces the
invasion-promoting effect of fibroblasts. These data suggest
that fibroblasts secrete some factors to activate
NF-κBmediated transcription of COX-2 in breast cancer cells
to enhance tumor progression.
However, several issues remain elusive. First, does PGE2
generated by COX-2-expressing cancer cells also affect gene
expression or behavior of stromal fibroblasts? Second,
do CAFs secrete small molecules (other than proteins
or peptides) to regulate cancer cell invasion? Finally,
can the importance of cancer-stroma interaction in cancer
progression be validated in clinical samples?
In this study, we address these questions and try to
clarify the underlying mechanism.
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 [
], 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.
Establishment of inducible COX-2-expression MCF-7 cell line
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
pTREmCherry-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 [
]. Metabolite identity was
predicted with Human Metabolome Database [
RNA extraction and quantitative reverse transcription-PCR analysis
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
2,3dioxygenase (TDO)-forward: 5′-GGGAACTACCTGCAT
TTGGA-3′; TDO-reverse: 5′-GTGCATCCGAGAAACA
ACCT-3′; IDO-forward: 5′-GCGCTGTTGGAAATAG
CTTC-3′; IDO-reverse: 5′-CAGGACGTCAAAGCACTG
AA-3′; E-cadherin-forward: 5′-CCTGGGACTCCACCTA
CAGA-3′; E-cadherin-reverse: 5′-GGATGAACACAGCG
TGAGAGA-3′; actin-forward: 5′-TGTTACCAACTGGG
ACGACA-3′; actin-reverse: 5′-GGGGTGTTGAAGGTCT
Immunoprecipitation and Western blot analysis
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-2phenylindole (DAPI), and the cell number in 15
randomly selected fields was counted under a microscope
(100×). Experiments were performed independently at
least 3 times.
Protein ubiquitination assay
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.
Immunofluorescent staining and confocal microscopy
MCF-7 cells were treated with or without 100 μM
kynurenine for 6 hours and fixed with 3.7% formaldehyde for
15 minutes at room temperature. Cells were washed twice
with PBS and permeabilized by 0.1% Triton X-100 solution
for 10 minutes. After permeabilization, cells were
incubated with 0.05% BSA solution to block nonspecific
binding. Anti-AhR mouse monoclonal antibody (1:80) or
anti-E-cadherin goat polyclonal antibody (1:250) was added
and incubated at room temperature for 1 hour. After
extensive washing, Alexas Fluro 594 anti-mouse IgG or
Alexas Fluro 488 anti-goat IgG was added and incubated
for another 1 hour. Cell nuclei were stained with DAPI
solution. Finally, coverslips were washed twice with PBS and
subsequently placed in mounting solution. The fluorescent
image was observed under a confocal microscope.
In vivo orthotopic animal study
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.CgFoxn1nu/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.
Patients and statistical analysis
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 [
], 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
diseasespecific 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.
COX-2-overexpressing breast cancer cells upregulated
IDO expression in co-cultured fibroblasts
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, [
(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
COX2-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-2overexpressing 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
COX2-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.
PGE2 transcriptionally elevated IDO expression in RMF-EG
fibroblasts through the EP4/STAT3 signaling pathway
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
]. Both STAT1 and STAT3 have been implicated in
the regulation of IDO expression [
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-2overexpressing breast cancer cells upregulate IDO
expression in fibroblasts through the PGE2/EP4/STAT3 pathway.
IDO-expressing fibroblasts enhanced the migration of breast cancer cells through downregulation of E-cadherin
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-2overexpressing MCF-7 cells (Figure 3B). The conditioned
medium of RMF-EG fibroblasts preincubated with
COX-2overexpressing 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 increased the degradation of E-cadherin in an
AhR- and Skp2-dependent manner
Kynurenine has been shown to be an endogenous
tumor-promoting ligand of the human AhR [
addition, AhR is involved in the degradation of sex
steroid receptors via a cullin 4B-dependent
ubiquitination pathway [
]. 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,
kynurenineincreased 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.
COX-2 expression in breast cancer and IDO expression in stromal fibroblasts predicted poor disease-specific and metastasis-free survival
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-2overexpressing 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).
COX-2 and IDO inhibitors suppressed growth of
COX-2-overexpressing breast tumors in vivo
The effect of COX-2 and IDO inhibitors was evaluated
in an orthotopic model. Inoculation of
MCF-7/RMF-EGor 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.
Previous studies demonstrated that IDO overexpression
increases the secretion of kynurenine to inhibit effect T
cells to promote immune escape and tumor progression
in various human cancers [
]. The expression of IDO
in cancer stroma has not been clarified. In addition, the
clinical significance of stromal IDO is unclear.
In this study, we provide evidence that
COX-2overexpressing breasts cancer cells may secrete PGE2 to
induce IDO expression and kynurenine production in
stromal fibroblasts. In addition, we show that kynurenine in
the coculture-conditioned medium is produced mainly
by CAFs because IDO is not induced by COX-2
overexpression in MCF-7 cells. An important upstream
regulator of IDO is interferon-γ. Yoshida et al. [
first reported that the pulmonary IDO was induced in
the mouse after intraperitoneal administration of bacterial
endotoxin or during in vivo virus infection, and this
induction was triggered by interferon-γ [
interferon-γ exhibits antitumor activity on various
cancers in vitro and in vivo, it is unlikely that
COX-2overexpressing cancer cells produce interferon-γ to
stimulate stromal IDO. For the first time, we show that
cancer cell-produced PGE2 transcriptionally
upregulates IDO expression through the EP4/STAT3 signaling
pathway. In vivo binding of STAT3 to IDO gene
promoter is confirmed by ChIP assay. Additionally,
knockdown of STAT3 totally abolishes EP4 agonist-induced
IDO expression. These data suggest that IDO is a
direct transcriptional target of STAT3.
An unresolved question is why PGE2 stimulates IDO
expression in stromal fibroblasts but not in breast cancer
cells, because both cell types express EP4 receptor [31
and data not shown]. We are aware that the binding of
STAT1 to IDO promoter is reduced by PGE2 (Additional
file 3: Figure S2); therefore, it is possible that the
expression level of STAT1 and STAT3 and the competition
between these two STATs may determine the response of
cells to PGE2 stimulation.
The concept of oncometabolite was established by
the studies that mutations of isocitrate dehydrogenase
1 (IDH1) and IDH2 generate a novel metabolite
2hydroxyglutarate (2-HG) that exhibits oncogenic
activity in acute myeloid leukemia and glioma [
Subsequently, 2-HG was shown to be a competitive
inhibitor of α-ketoglutarate-dependent dioxygenases and inhibits
histone demethylases like Tet methylcytosine dioxygenase
2 (TET2) to change promoter methylation and gene
]. Kynurenine represents another
oncometabolite, which acts as an immunosuppressor to create
a favorable microenvironment for tumor formation
and metastasis [
]. A recent study demonstrated that
the tryptophan catabolism enzyme TDO is
overexpressed in human brain tumors, and elevated secretion
of kynurenine promotes cell migration via an
AhRdependent pathway [
However, the underlying mechanism by which
kynurenine increases cell motility is still unclear. After
screening of the EMT markers, we found that E-cadherin is
decreased in kynurenine-treated breast cancer cells, and
AhR is involved in this process. AhR has been shown
to integrate as a component of a novel Cul4B ubiquitin
Age (years) <60 years
Primary tumor (T) T1
Nodal status (N) N0
Histologic grade Grade I
Cox-2 expression Low Exp (<medium)
(Tumor) High Exp (≧medium)
Ido-1 expression Low Exp (<medium)
(CAF) High Exp (≧medium)
Bold figures, Statistically significant.
E3 ligase complex and participated in the degradation
of sex steroid receptors [
]. We demonstrated that
kynurenine increases the interaction between AhR and
E-cadherin, and the AhR/E-cadherin complex also contains
Skp2, an F-box protein of SCF E3 ligase. The formation
of the E-cadherin/AhR/Skp2 complex and ubiquitination
of E-cadherin induced by kynurenine is also detectable in
A549 cells, indicating a general mechanism of
kynurenineinduced proteolysis of E-cadherin in different cancer cells.
Our results provide a novel oncometabolite function of
kynurenine to enhance cancer cell migration by degrading
The clinical validation of tumor COX-2 and stromal
IDO in this study is important to verify the
cancerstroma interplay in cancer progression. Many
histopathologic studies investigated the expression of two specific
genes in the epithelial components of tumor tissues to
show their association and to demonstrate the vertical
regulation of these two genes. The correlation and
clinical significance of genes separately expressed in tumor
and stroma have received little attention.
However, the gene signatures in CAFs may provide
more information than originally thought. West et al.
] first classified two stromal gene signature from
tumors with solitary fibrous tumor (SFT) and desmoids-type
fibromatosis (DTF) features and showed that patients with
the expression of DTF had a favorable clinical outcome.
Their subsequent study by using public databases and
immunohistochemical approaches suggested that DTF
fibroblast signature is a common tumor stroma signature
in different types of cancers [
]. Mercier et al. [
identified a hyperproliferative gene signature in CAFs and found
that breast cancer patients with this signature had a poor
prognosis with tamoxifen monotherapy and a great
reduction in recurrence-free survival [
]. By using a mouse
model of squamous skin carcinogenesis, Erez [
demonstrated that carcinoma cells could educate CAFs to
express proinflammatory genes to promote macrophage
recruitment, neovascularization, and tumor growth.
Additionally, this gene signature was also evident in
mammary and pancreatic tumors in mice and in human cancers.
By using metabolomics, molecular, and pathological
approaches, we revealed that induction of stromal IDO
by COX-2-overexpressing breast cancer cells promotes
tumor progression and predicts poor patient survival.
Results of our animal study also clearly demonstrate
the anticancer effect of COX-2 and IDO inhibitor on
COX-2-overexpressing breast cancer in vivo. A
combination of IDO and COX-2 inhibitor exhibits a more
obvious effect on the inhibition of tumor growth. However,
we did not find an additive effect. This can be because
(a) the number of animals in each group is small, and
(b) inhibition of COX-2 in cancer cells will attenuate
stromal IDO expression, which reduces the anticancer
activity of IDO inhibitor. Additional experiments are
needed to clarify this issue.
By using a metabolomics approach, we identified potential
oncometabolites involved in the crosstalk between
COX-2overexpressing breast cancer cells and fibroblasts.
Molecular study elucidates the underlying mechanism by which
this cancer/stroma interplay via COX-2 and IDO promotes
tumor progression. In addition, pathological investigation
validates the importance of cancer COX-2 and stromal
IDO in the prediction of the patient’s survival.
Simultaneous targeting of COX-2 and IDO may be a new strategy
for breast cancer treatment.
Additional file 1: Supplementary materials and methods.
Additional file 2: Figure S1. PGE2 stimulated IDO promoter activity.
Different IDO promoter constructs were transfected into MCF-7 cells and
stimulated by PGE2. Promoter assay indicated that PGE2 activated IDO via
the −1140/-844 promoter region.
Additional file 3: Figure S2. In vivo binding of STAT3 on IDO gene
promoter in EMF-EG fibroblasts and its regulation by co-culture of
COX-2-overexpressing MCF7 cells. ChIP assay demonstrated that the
binding of STAT3 to IDO promoter was increased, whereas the binding of
STAT1 was reduced in EMF-EG fibroblasts after co-culture with
COX-2-overexpressing MCF-7 cells.
Additional file 4: Figure S3. Kynurenine induced the formation of
E-cadherin/AhR/Skp2 complex in A549 lung cancer cells. A549 cells were
treated without (−) or with (+) kynurenine, and the interaction between
E-cadherin and AhR or Skp2 was studied by immunoprecipitation and
Additional file 5: Figure S4. IDO expression in cancer-associated
fibroblasts (CAFs) was increased in COX-2-overexpressing breast cancer.
2-HG: 2-hydroxyglutarate; 3′4′-DMF: 3′4′-dimethoxyflavone; 3-MC:
3′-methylcholanthrene; AhR: aryl hydrocarbon receptor; CAF: cancer-associated
fibroblast; CXCL12: chemokine (C-X-C motif) ligand 12; CXCL14: chemokine
(C-X-C motif) ligand 14; DAPI: 4′,6-diamidino-2-phenylindole; DTF: desmoid-type
fibromatosis; ELISA: enzyme-linked immunosorbent assay; EMT:
epithelial-mesenchymal transition; EP4: prostaglandin E receptor 4;
IDH: isocitrate dehydrogenase; IDO: indoleamine 2,3-dioxygenase; MMP: matrix
metalloproteinase; NF-κB: nuclear factor kappa-light-chain-enhancer of activated
B cells; PGE2: prostaglandin E2; SDF-1: stromal cell-derived factor 1; SFT: solitary
fibrous tumor; shRNA: short-hairpin RNA; Skp2: S-phase kinase-associated
protein 2; STAT3: signal transducer and activator of transcription 3; TET2: Tet
methylcytosine dioxygenase 2; UPLC: ultraperformance liquid chromatography.
The authors declare that they have no competing interests.
JY conducted cell culture, biochemical, and molecular biology assays and
prepared the draft of the manuscript. CF did IHC study and pathological analysis
and helped to draft the manuscript. CC performed metabolomics study and data
analysis and helped to draft the manuscript. KK participated in the design of the
study and helped to draft the manuscript. MF conceived of the study and
participated in the preparation of primary cancer-associated fibroblasts. WC
conceived of the study and participated in the design of the study and wrote the
manuscript. All authors read and approved the final manuscript.
We deeply thank Dr. Wu KK for his continuous support and encouragement. We
also thank Ms. Pei-Yung Nien for the isolation of primary CAFs and Dr. Delphine
Allorge and Dr. Marion Soichot for providing human IDO promoter. This study
was supported by grants from National Science Council (NSC 101-2321-B-400-003
and NSC 102-2321-B-400-003) and MOHW103-TD-B-111-05 from Excellence for
Cancer Research Center Grant, the Ministry of Health and Welfare.
Kaohsiung 807, Taiwan. 5Department of Surgery, Kaohsiung Municipal
Ta-Tung Hospital, Kaohsiung 807, Taiwan. 6Cancer Center, Kaohsiung Medical
University Hospital, Kaohsiung 807, Taiwan. 7Graduate Institute of Medicine,
College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
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