Accepted Manuscript
Title: Role of Chemokine Receptor CXCR7 in Bladder CancerProgression
Authors: Mingang Hao, Jianghua Zheng, Kailin Hou, JinglongWang, Xiaosong Chen, Xiaojiong Lu, Junjie Bo, Chen Xu,Kunwei Shen, Jianhua Wang
PII: S0006-2952(12)00265-1DOI: doi:10.1016/j.bcp.2012.04.007Reference: BCP 11261
To appear in: BCP
Received date: 6-3-2012Revised date: 6-4-2012Accepted date: 6-4-2012
Please cite this article as: Hao M, Zheng J, Hou K, Wang J, Chen X, Lu X, Bo J, Xu C,Shen K, Wang J, Role of Chemokine Receptor CXCR7 in Bladder Cancer Progression,Biochemical Pharmacology (2010), doi:10.1016/j.bcp.2012.04.007
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Role of Chemokine Receptor CXCR7 in Bladder Cancer
Progression
Mingang Hao# a, Jianghua Zheng# a, Kailin Hou # c, Jinglong Wanga, Xiaosong
Chenb, Xiaojiong Lud, Junjie Boc, Chen Xue, Kunwei Shenb, Jianhua Wanga*
a Department of Biochemistry and Molecular & Cell Biology, Shanghai Jiao Tong
University School of Medicine, Shanghai, 200025, China.
b Shanghai Ruijin Hospital, Comprehensive Breast Health Center, Shanghai, 200025,
China.
c Department of Urology, Shanghai Renji Hospital, Shanghai, 200001, China.
d Department of Surgery, Division of Trauma/Surgical Critical Care/Burns, Medical
Center (Hillcrest), University of California, San Diego MC8896 UN.
e Department of Histology and Embryology, Shanghai Jiao Tong University School of
Medicine, 200025, China
#M Hao, J Zheng, and K Hou contributed equally to this work.
*Corresponding author: Jianhua Wang, PhD, Shanghai Jiao Tong University School
of Medicine, Shanghai, 200025, China. Phone: 021-54660871; Fax: 021-63842157;
E-mail:[email protected].
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Abstract
Bladder cancer is one of the most common tumors of the genitourinary tract; however,
the molecular events underlying growth and invasion of the tumor remain unclear.
Here, role of the CXCR7 receptor in bladder cancer was further explored. CXCR7
protein expression was examined using high-density tissue microarrays. Expression of
CXCR7 showed strong epithelial staining that correlated with bladder cancer
progression. In vitro and in vivo studies in bladder cancer cell lines suggested that
alterations in CXCR7 expression were associated with the activities of proliferation,
apoptosis, migration, invasion, angiogenesis and tumor growth. Moreover, CXCR7
expression was able to regulate expression of the proangiogenic factors IL-8 or VEGF,
which may involve in the regulation of tumor angiogenesis. Finally, we found that
signaling by the CXCR7 in bladder cancer cells activates AKT, ERK and STAT3
pathways. The AKT and ERK pathways may reciprocally regulate, which are
responsible for in vitro and in vivo epithelial to mesenchymal transition (EMT) process of
bladder cancer. Simultaneously targeting the two pathways by using U0126 and
LY294002 inhibitors or using CCX733, a selective CXCR7 antagonist drastically
reduced CXCR7-induced EMT process.
Taken together, our data show for the first time that CXCR7 plays a role in the
development of bladder cancer. Targeting CXCR7 or its downstream-activated AKT
and ERK pathways may prove beneficial to prevent metastasis and provide a more
effective therapeutic strategy for bladder cancer.
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1. Introduction
Bladder cancer is the ninth most common type of cancer worldwide, which affects
three times as many men as women. Each year, about 360,000 new cases of bladder
cancer are expected, and about 150,000 people will die of this disease in the world [1].
It is the fifth most common type of cancer in the China-the fourth most common in
men and the ninth in women [2]. The clinical course of bladder cancer carries a broad
spectrum of aggressiveness and risk. High-grade non–muscle-invasive cancers
frequently progress and muscle-invasive cancers are often lethal and have the highest
recurrence rate of any malignancy [1, 3]. Intriguingly, emerging evidence suggests
that chemokines are likely to play a crucial role in mediating the continuous series of
events leading to tumor growth and metastasis [3-8]. Therefore, it is important to
investigate chemokine signaling in bladder cancer to get a better understanding of the
underlying mechanisms leading to tumor invasion and metastasis, and to develop
prognostic and therapeutic strategies in this disease.
Over the past several years, there is emerging evidence that multiple pairs of
chemokines and their receptors play critical roles in cancer progression [4, 5, 7]. Our
group and others have shown that CXCL12 and its receptor CXCR4 are critical
elements in growth and metastasis of prostate cancer (PCa) to tissues that produce
large quantities of CXCL12 [6, 9]. More recently, CXCL12 was shown to bind with
high affinity to the orphan receptor CXCR7. CXCR7 was originally cloned on the
basis of its homology with conserved domains of G protein-coupled receptors [10, 11].
Combined phylogenetic and chromosomal location studies suggest that CXCR7
represents a chemokine receptor structurally close to CXC receptors [12], pointing to
CXC chemokines as potential ligands. CXCR7 shares homology with the viral gene
ORF74 encoding a chemokine receptor, which suggests that it may signal
constitutively in the absence of ligand [13]. Like CXCR4, CXCR7 serves as a co-
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receptor for some human and simian immunodeficiency virus strains [14] . In the
vasculature, the expression of CXCR7 is elevated in endothelial cells associated with
tumors, and overexpression of CXCR7 in NIH 3T3 cells strongly supports a role for
the receptor in tumorigenesis [14]. More recently, CXCR7 expression has been shown
to be elevated in endothelial cells associated with tumors [15]. Membrane associated
CXCR7 is expressed on many tumor cell lines, on activated endothelial cells, and on
fetal liver cells [16]. CXCR7 is expressed by the placenta [17] as well as in the
vascular endothelium [18, 19]. Miao et al. [18] further confirmed a critical role for
CXCR7 in tumor vascular formation, angiogenesis, and promotion of the growth of
breast and lung cancer in vivo. These characteristics suggest that like CXCR4,
CXCR7 plays a role in regulating immunity, angiogenesis, stem cell trafficking, and
mediating organ-specific metastases of cancer. However, the role of CXCR7 in
bladder cancer still remains unsettled.
Importantly, growing evidence has suggested that CXCR7 functions as a decoy
receptor, which does not activate Gi pathways of a chemokine receptor that may result
in GTP hydrolysis or calcium mobilization [16]. Debate has still arisen whether
CXCR7 functions like GPCR mediating the signal transduction process [20]. More
recently, it has been demonstrated that CXCR7 interacts with β-arrestin in a ligand-
dependent manner [20-23]. CXCR7 can signal through β-arrestin and act as an
endogenous β-arrestin-biased receptor, which suggests that other receptors that are
currently thought to be orphans or decoys may also signal through non-G-protein-
mediated mechanisms [20].
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2. Materials and methods
2.1. Cell culture
Human bladder cancer cell lines with different grade T24 (grade 3 bladder cancer),
J82 (grade 3 bladder cancer) and RT4 (grade 1 bladder cancer) were purchased from
American Type Culture Collection, which have been widely used by other groups as a
model for bladder cancer. These cell lines were cultured in DMEM, RPMI 1640, and
McCoy's 5a Medium containing fetal bovine serum (FBS) with antibiotics (Invitrogen
Corp., Carlsbad, CA), respectively. Additional methodologies are provided in the
Supplementary sections.
2.2. Tissue microarrays and immunohistochemical staining
High-density tissue microarrays were constructed by Shanxi Chaoying Biotechnology
Co.,Ltd. (Catalog No: CC12-11, Xian, China) with clinical samples obtained from a
cohort of 78 patients. The clinical samples used to perform in the project have been
approved by Shanghai Jiao Tong University School of Medicine Ethical Committee.
The method of quantitative analysis was previously described [24]. Additional
methodologies are provided in the Supplementary sections.
2.3. Construction of lentiviral vectors
Stable knock-down of CXCR7 in RT4 cells was achieved by expression of shRNA
from lentivirus vector pLLU2G-shGremlin under the control of CMV promoter for
stable expression (Cyagen Bioscience). Lentivirus vector pLLU2G-shGremlin was
purchased from Cyagen Biosciences Inc. (Cyagen Bioscience). Three pairs of
annealed DNA oligonucleotides were inserted between HpaI and XhoI restriction sites
according to common protocol. Additional methodologies are provided in the
Supplementary sections.
2.4. RNA extraction and RT-PCR
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Total RNA in T24, J82 and RT4 cells were extracted using Trizol (Invitrogen,
Carlsbad, CA). RT-PCR was performed using reverse transcriptase cDNA synthesis
kit (Fermentas, St Leon-Rot, Germany) according to the manufacturer’s protocol. 1
μg of total RNA were reversely transcribed into cDNA and equal volume of cDNA
were used as PCR template using specific primers: CXCR7, Genbank no.:
NM_020311, (sense) 5’-AGGAAGTAGAAGACAGCGATAATGG-3’, (anti-sense)
5’-TATGACACGCACTGCTACATCTTG-3’; CXCL11, Genbank no.: NM_005409,
(sense) 5’-AGCCTCCATAATGTACCCAA-3’, (anti-sense) 5’-
GCACTTTGTAAACTCCGATG-3’; CXCL12, Genbank no.: NM_199168, (sense)
5’- GAGCCAACGTCAAGCATCT-3’, (anti-sense) 5’-
CGGGTCAATGCACACTTGTC-3’; GAPDH, Genbank no.: NM_002046, (sense)
5’-TCACCATCTTCCAGGAGCGAGA-3’, (anti-sense) 5’-
GCAGGAGGCATTGCTGATGATC-3’. CXCR7, CXCL11, CXCL12 and GAPDH
were amplified by 30 cycles at 95℃ for 20 s, 60℃ for 60 s and 68℃ for 30 s (NEB,
Ipswich, MA).
2.5. siRNAs of CXCR4 and transfection
Transient knock-down of CXCR4 by transduction of siRNA duplexes targeting
CXCR4 mRNA was achieved as previously described [25]. siRNA duplexes were
purchased from RiboBio (RiboBio, Guangzhou, China) and the most effective
duplexes are: (sense), 5’-UAAAAUCUUCCUGCCCACCdTdT-3’, (anti-sense), 5’-
GGUGGGCAGGAAGAUUUUAdTdT-3’. The scramble siRNA duplexes were used
as control. The siRNA duplexes were transduced into RT4 and J82 cells at a final
concentration of 120 nM using Lipofectamine2000 (Invitrogen, Carlsbad, CA).
2.6. Flow cytometry analyses
To determine total cell surface CXCR7 and CXCR4 expression, 105 cells were
incubated at room temperature for 30 min with 10 μL of nonspecific isotype-matched
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controls, mouse to human IgG (R&D Systems, Minneapolis, MN) and 10 μL of
mouse� monoclonal antibodies conjugated PE fluorochrome (anti-human CXCR7,
11G8) (R&D Systems, Minneapolis, MN) and APC fluorochrome (anti-human
CXCR4, 12G5) (BD Biosciences, San Diego, CA). Unbound antibodies were
removed by washing the cells twice in phosphate-buffered saline buffer. Cells were
analyzed by FACScan (BD Biosciences) and the data were analyzed with CellQuest
software (BD Biosciences).
2.7. Proliferation assay
After a 24-h serum withdrawal, bladder cancer cells were digested and plated at 1×104
cells/well into triplicate 96-well flat-bottomed cell culture plates in 0.1ml complete
culture medium. Additional methodologies are provided in the Supplementary
sections.
2.8. Cell migration and invasion
Wound healing assay was used to assess cell migration. Cell invasion was examined
using a reconstituted extracellular matrix membrane (BD Biosciences, San Jose, CA).
Additional methodologies are provided in the Supplementary sections.
2.9. Western blots
Cells were cultured in 3.5 cm diameter plates (80-90% confluence), and washed by
PBS, and then serum-starved for overnight. The cells were then stimulated with 200
ng/ml CXCL12 (R&D Systems, Minneapolis, MN) or not for 10 min at 37°C and
lysed for 10 min on ice in RIPA buffer (Thermo Scientific, Waltham, MA) containing
an anti-protease mix (Roche, Germany), and protein concentration was measured by
BCA assay (Thermo Scientific, Waltham, MA). Additional methodologies are
provided in the Supplementary sections.
2.10. ELISA
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Antibody sandwich ELISAs were used to evaluate IL-8, and VEGF levels in the PCa
cell conditioned medium (CM) (R&D Systems, Minneapolis, MN) as previously
described.
2.11. Cytokine antibody arrays
The expression of 20 cytokines was evaluated using human angiogenesis antibody
arrays (Catalog No: AAH-ANG-1) (Ray-Biotech, Norcross, GA). The membranes
were exposed to blocking buffer for 1 h at 25°C, and incubated with CM or control
medium up to 2 h at 25°C. After washing three times, the arrays were processed with
biotin-conjugated angiogenesis antibody mix and strepavidin-HRP conjugate
according to the manufacturer protocol.
2.12. Endothelial sprout formation assays
Growth factor reduced basement membranes were placed into 4-chamber slides
(MatrigelTM 125 µl/chamber; BD Biosciences, San Diego, CA) and 0.8 × 104
endothelial cells were added on top. Additional methodologies are provided in the
Supplementary sections.
2.13. cDNA microarray analysis
Illumina Human HT12 v 3 Expression BeadChips (Illumina) were used in this study.
This BeadChip targets > 25,000 genes with > 48,000 probes derived from the RefSeq
(Build 36.2, rel22) and UniGene (Build 99) databases.
Additional methodologies are provided in the Supplementary sections.
2.14. Subcutaneous tumor growth
All experimental animal procedures were performed in compliance of the institutional
ethical requirements and were approved by the Shanghai Jiao-Tong University School
of Medicine Committee for the Use and Care of Animals. Additional methodologies
are provided in the Supplementary sections.
2.15. Immunohistochemistry
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For immunostaining with CD31, tissue sections were blocked with Sniper for 5 min
and incubated overnight at 4 °C with 28 mg/ml rabbit anti-human CD31 antibody
(Dako North America Inc., Carpinteria, CA) diluted in PBS. Additional
methodologies are provided in the Supplementary sections.
2.16. Statistics
Numerical data are expressed as means ± standard deviation. Statistical differences
between the means for the different groups were evaluated with Instat 4.0 (GraphPAD
software) using one-way analysis of variance (ANOVA) with the level of significance
at p < 0.05. Where indicated, a Krusal-Wallis test and Dunn’s multiple comparisons
tests were utilized with the level of significance set at p < 0.05.
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3. Results
3.1. Expression of CXCR7 in bladder cancer
To explore the role of CXCR7 in bladder cancer, high density tissue microarrays
were stained with an anti-human CXCR7 from clinical samples obtained from a
cohort of 78 patients. Representative images are shown in Fig. 1A, indicating that
expression of CXCR7 in bladder cancer tissues (grade I, II, III) is markedly higher
than the normal tissues. High grade II and III also indicated stronger staining than low
grade I (Fig. 1A). By quantitative analysis, these findings suggested that CXCR7
expression increases with increasing tumor grade (Fig. 1B). Moreover, we recently
showed by Kaplan-Meier analysis that in 148 patients followed up for 2-95 months,
overexpression of CXCR7 is significantly associated with shorter recurrence-free
survival (RFS) rate (log -rank, 3.256; p = 0.002) [26]. Therefore, these results
demonstrate that CXCR7 expression is correlated with bladder cancer progression.
3.2. CXCR7 modulates phenotype of bladder cancer cells.
RT-PCR and FACS analyses of CXCR7 showed higher expression in the RT4
compared with J82, T24 cell lines (Fig. 2A, up and bottom). Next CXCR7 expression
was modulated by overexpressing the receptor in J82, T24 cells (noted as J82CXCR7,
T24CXCR7) or by reducing its expression by shRNA (noted as RT4shCXCR7). After clone
selection, individual clones were pooled and evaluated by FACS for CXCR7
expression in these cell lines (Fig. 2B). As shown in Fig. 2C, overexpression of
CXCR7 increased the basal proliferation rates of the J82 and T24 cells in 4 day,
whereas reducing the receptor expression in RT4 cells decreased the effects.
One possible explanation of the increased proliferation observed after transfection
with CXCR7 is that the receptor may protect the cell lines from apoptosis. The loss of
cellular membrane integrity as a reflection of cells undergoing apoptosis was
therefore determined by staining the cells for annexin-V. As shown in supplemental
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Fig. 1, CXCR7 overexpression decreased the apoptotic fraction of cells in culture for
both J82 and T24 cells compared with the respective controls (8.9 % versus 4.5 % and
13.8 % versus 12.5 %). The total number of dead or dying cells (double-positive for
PI and annexin-V) (10.7 % J82CXCR7 versus 6.1 % J82control ; 20.0 % T24CXCR7 versus
16.0 % T24control) was less as well.
The ability of CXCR7 to regulate migration was assessed by scratch healing assay.
The results showed that the CXCR7- transfected cells nearly closed the wound at 36 h
for J82 cells or 12h for T24 cells after scratch, whereas the respective control cells
were unable to heal the wound at the respective time. The mean wound distances of
the CXCR7-transfected cells and the control cells at 36 h for J82 cells or 12h for T24
cells were significantly different (165.28 ± 6.18 m vs 435.231 ± 53.06 m; 8.17 ±
6.41 m vs 189.31 ± 24.67 m p < 0.001) (Fig. 2D). The results indicate that CXCR7
expression is capable of inducing migration of bladder cancer cells.
Once individual tumor cells are bound to the endothelium, they must invade through
the extracellular matrix to establish a metastasis. The ability of CXCR7 to regulate
invasion was next studied using reconstituted extracellular matrices in porous culture
chambers (Matrigel; Beckman Coulter Labware). J82 cells with overexpression of
CXCR7 resulted in markedly higher invasive ability compared with the control (Fig.
2E) in the presence of CXCL12, but not in CXCR7-overexpressing T24 cells. The
possibility is that CXCR4 expression is absent in T24 cells, which is responsible for
insignificant invasion (Supplemental Fig. 2). In contrast, reducing the expression of
CXCR7 decreased invasive abilities of J82 cells in the presence of CXCL12 (Fig. 2E).
To determine whether CXCR7 and CXCR4 reciprocally regulate each other in
bladder cancer, similar studies were performed in cells with altered CXCR7 levels. As
shown in supplemental Fig. 2, alterations of CXCR7 expression did not significantly
regulate CXCR4 levels in these bladder cell lines as previously reported in PCa [19].
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In contrast, knockdown of CXCR4 expression in J82 and RT4 cells resulted in
enhanced levels of CXCR7 as compared with the respective controls (Supplemental
Fig. 2).
3.3. Effect of altered CXCR7 expression on cytokine secretion.
The growth of new blood vessels (angiogenesis) within tumors by altered cytokine
secretion is essential for tumor growth, maintenance, and metastasis [7-9]. Based on
these findings, we hypothesized that modulated CXCR7 expression is potentially
linked to regulating expression of angiogenic growth factors in bladder cancer cells.
To test this possibility, antibody arrays targeting angiogenic cytokines were used. As
shown in Fig. 3A, high levels of VEGF and IL-8 were commonly presented in the CM
derived from T24 and J82 cells with overexpression of CXCR7 compared with the
respective controls. To further explore the proangiogenic signals regulated by CXCR7,
CM derived from the various CXCR7 transfectants were analyzed by ELISA. Fig. 3B
indicated that IL-8 and VEGF levels in T24 and J82 cells overexpressing CXCR7
were higher than the control cells, consistent with our antibody array data. In contrast,
shRNA targeting of CXCR7 expression in RT4 cells resulted in significant reductions
in the levels of IL-8 and VEGF compared with the control cells.
To explore whether CXCR7 expression is biologically relevant to vascular
recruitment by bladder cancer cells, human endothelial cell sprout formation was used
as in vitro assay measuring proangiogenic activity. Shown in Fig. 3C, little or no
sprout formation occurred in the absence of external stimuli applied to endothelial
cells alone in vitro. Co-culture of the endothelial cells with either the CM derived
from J82 or T24 control cells stimulated robust vascular sprout formation (Fig. 3C).
However, overexpression of CXCR7 in both cells dramatically increased blood vessel
sprout formation (Fig. 3C) compared with their respective controls. In contrast,
reduced expression of CXCR7 in RT4 cells decreased vessel formation relative to the
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respective control (Fig. 3C).
3.4. CXCR7 promotes EMT of bladder cancer cells
As shown in Supplementary Fig. 3, altering expression of CXCR7 induced the
morphological appearance similar to EMT or MET phenotype, suggesting that
CXCR7 may be associated with EMT or MET in bladder cancer progression. To test
the possibility, we assessed the expression of EMT marker proteins as previously
reported [27]. The results showed that although E-cadherin, a universal epithelial
marker, is absent in J82 and T24 cells, overexpression of CXCR7 in both cells
induced up-regulation of N-cadherin and -catenin, two mesenchymal markers. In
contrast, CXCR7 shRNA-targeted RT4 cells increased E-cadherin expression
coincided with the reduction of N-cadherin and -catenin expression (Fig. 4A).
To explore potential cellular pathways of EMT induced by CXCR7, we analyzed
the genome-wide transcriptome profile of CXCR7 shRNA-targeted RT4 and the
control cells by Illumina Human HT12 v 3 Expression BeadChips (Illumina).
According to fold-change (X2.0) screening between RT4shicontrol and RT4shCXCR7 cells,
we found 310 up-regulated genes and 267 down-regulated genes (Supplementary
Table 1). As shown in Fig. 4B, 47 cancer-associated genes for cluster mapping on the
MeV microarray analysis platform (www.tm4.org/mev.heml). We also identified
genes related to molecular function of EMT and picked up the top 9 gene sets that
overlapped with different function-clusters for exhibition (Fig. 4C).
3.5. EMT induced by CXCR7 is associated with the activated AKT, ERK and
STAT3 pathways.
The above microarray data imply that CXCR7 signaling may be able to activate some
pathways, which acts as a player in EMT process of bladder cancer. As shown in Fig.
5A, overexpression of CXCR7 in J82 and T24 cells induced marked phosphorylation
of Akt at Thr-308 site, but not at Ser-473 site (Fig. 5B); In contrast, knockdown of
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CXCR7 in RT4 cells reduced intense phosphorylation of Akt (Thr-308). Similar
induced trend were observed in these cells that altering CXCR7 expression is
responsible for activating ERK and STAT3 (Tyr-705) pathways (Fig. 5A).
Interestingly, we noted that targeting the ERK pathway by using U0126 in CXCR7-
expressing J82 cells can activate AKT pathway. Yet, targeting the AKT pathway by
using LY294002 can also activate ERK pathway (Fig. 5B). The results imply that
AKT and ERK signaling by CXCR7 may reciprocally regulate in bladder cancer cells.
EMT has been noted as a critical process that will contribute to tumor invasive
phenotypes, which is associated with multiple activated pathways, including AKT and
ERK signaling et al (25). Fig. 5B showed that activating AKT and ERK pathways by
CXCR7 can both significantly up-regulated expression of -catenin and N-cadherin.
In this case, simultaneously targeting the two pathways by using U0126 and
LY294002 can greatly reduce expression of -catenin and N-cadherin compared with
inhibiting the one pathway alone, however. Similarly, using CCX733, a selective
CXCR7 antagonist, drastically reduced CXCR7-induced expression of -catenin and
N-cadherin compared with another antagonist CCX771 and negative control CCX704,
followed by inhibiting phosphorylation of Akt (Thr-308) and ERK signaling. Taken
together, the results imply that activating AKT and ERK pathways by CXCR7 are
responsible for in vitro EMT process of bladder cancer cells. However, whether the
activated STAT3 pathway by CXCR7 also involved in EMT process is warranty
needed to further investigate.
3.6. Effects of CXCR7 expression on tumor growth, angiogenesis and EMT in
vivo.
To confirm that CXCR7 plays a role in tumor growth, SCID mice were implanted s.c.
with cells engineered to overexpress CXCR7 level. As shown in Fig. 6A, B, tumor
burdens generated from CXCR7-overexpressing J82 cells were much larger than the
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tumors generated by the transfected control cells (535 mm3 versus 196 mm3).
To address directly whether CXCR7 was able to play a role in tumor angiogenesis,
the tumors were stained with an antibody to CD31, a specific marker for vascular
endothelial cells. Overexpression of CXCR7 in J82 cells resulted in more large and
abundant blood vessel formation than the tumors generated by the control transfected
cells (Fig. 6C) (26.2 1.6 versus 9.8 2.6 per high powered field, respectively).
Given that CXCR7 functions as a player in EMT process of bladder cancer in vitro by
activating AKT and ERK pathways, we next assay whether the effects may contribute
to tumor growth in vivo. Fig. 6C indicated that, CXCR7-overexpressing J82 in tumor
tissues expressed higher phosphorylated AKT (Thr-308) and ERK levels relative to
the respective controls. In this case, expressions of N-cadherin and -catenin are
significantly increased in CXCR7-overexpressing J82 tumor compared with the
controls. Taken together, these data suggest that activation of AKT and ERK
signaling by CXCR7 in J82 cells indeed play a role in EMT process of bladder cancer.
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4. Discussion
CXCR7, formerly an orphan receptor, has recently been identified as a chemokine
receptor for CXCL12, where it is expressed by many human cell lines, vascular
endothelial cells, and in rodent brain, kidney, lung, heart, spleen, pancreas, small
intestine, blood, colon, and blood vessels [11-13]. In addition, recent findings suggest
that expression of CXCR7 is elevated in endothelial cells associated with tumors [28].
Shimizu et al, recently found that intracellular CXCR7 expression is mainly located
into cytoplasm of human adult brain and differentiated neurons under normal and
pathological conditions [29]. We recently demonstrated that CXCR7 expression is
correlated with PCa metastasis and progression and suggest potential targets for
therapeutic intervention [19, 30]. However, to our knowledge, there is no report on
the expression and functional contribution of CXCR7 in bladder cancer development.
Here, we found that CXCR7 expression in clinical patients is correlated with bladder
cancer progression. Notably, Fig2.A indicated that CXCR7 expression in RT4, a
grade 1 bladder cancer cell line, is higher than T24 and J82, grade3 bladder cancer
cell lines, suggesting that these cell lines as the bladder cancer model may not be
perfect representative of phenotype of clinical patients. The potential reason is that
these cell populations established from bladder cancer patients are heterogeneous,
comprising different genomic characteristics and different abilities to metastasize to
distant secondary sites. By using in vivo selection in SCID mice, it may prove
effective in isolating highly representative of phenotype from the original bladder
cancer [31]. However, In vitro and in vivo studies in bladder cancer cell lines showed
that alterations in CXCR7 expression were associated with the activities of
proliferation, apoptosis, migration, invasion and tumor growth. This may correlate
with the signaling of CXCR7 through activation of the AKT and ERK pathways. We
also observed that CXCR7 appears to regulate blood vessel formation in bladder
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cancer progression as it was noted that both IL-8 and VEGF levels were altered in
response to changes in CXCR7 expression. These findings were in keeping with the
number of blood vessels formed by the tumors in vivo. IL-8 and VEGF have been
shown to be important factors involved in the development of tumor blood supply in
the progression of solid tumors upon activating AKT and ERK pathways [32, 33].
Together, our results suggest that CXCR7 may act as a player in the disease.
CXCR7 was originally cloned on the basis of its homology with conserved domains
of G protein-coupled receptors [10, 11] as an orphan receptor. Currently, it is accepted
that CXCR7 has two ligands, the chemokines CXCL12 and CXCL11, which also bind
to the chemokine receptor CXCR3 [10, 11]. CXCL12 mRNA is highly expressed in
lymph nodes, lung, liver and bone marrow, and multiple tumor cells [10, 11], whereas
CXCL11 is primarily expressed in peripheral blood leukocytes, pancreas, liver,
thymus, spleen and lung and less expressed in small intestine, placenta and prostate
[10, 11]. Recently, Singh RK, et al. showed that addition of CXCL12 and CXCL11 to
PCa cells did not affect cell proliferation. Overexpression of CXCR7 in normal
prostate cells increased their proliferation in a manner associated with increased levels
of p-EGFR and p-ERK1/2, which suggest that CXCL12 or and CXCL11 may be
nonessential to induce mitogentic response of CXCR7-overexpressing cells [34].
Indeed, we found that overexpression of CXCR7 in J82 and T24 cells only enhanced
CXCL11 gene expression, but not altered CXCL12. Administration of different
concentration of CXCL11 to these cells didn’t induce a significant proliferating
response (Supplementary Fig 4).
Notably, since CXCR7 has been confirmed as a second receptor for CXCL12,
debates are still arisen whether CXCR7 functions like GPCR mediating the signal
transduction process. We and other groups indicated that CXCL12 engagement to
CXCR7 can indeed transmit a range of cellular responses, such as, activation of ERK
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and AKT pathways, cell survival, proliferation, and invasion et al, and finally
chemotaxis of CXCR4-negative cells [35-37]. However, growing evidence has
suggested that CXCR7 function as a decoy receptor, which also constitutively
interacts with inactive G proteins but dramatically fails to activate them and mobilize
intracellular calcium mobilization once engaged by CXCL12 [10, 16, 18, 19].
Recently, Levoye et al proposed that chemotaxis mediated by CXCR7 may involve
the ability of CXCR7 to modulate CXCR4 signaling by scavenging CXCL12,
thereby modifying the chemokine concentration in the extracellular environment [38].
These findings raise one potential mechanism that CXCR7 can regulate CXCR4
activity through heterodimer formation [38, 39]. Moreover, Decaillot et al [40] further
demonstrate that CXCR7, which cannot signal directly through G protein-linked
pathways, can nevertheless affect cellular signaling networks by forming a
heterogenic complex with CXCR4. The CXCR4-CXCR7 heterodimer complex
recruits -arrestin, which results in preferential activation of -arrestin-linked
signaling pathways over canonical G protein pathways. It can potentiate cell
proliferative kinase pathways, including p38MAPK, SAPK and ERK1/2 activation
leading to increased cell migration of CXCR4-expressing breast cancer cells [40].
Here, we found that signaling by the CXCR7 in bladder cancer cells simultaneously
activates AKT and ERK signaling. Interestingly, in CXCR7-expressing J82 cells,
targeting the ERK pathway by using U0126 can activate AKT pathway; Yet, targeting
the AKT pathway by using LY294002 can also activate ERK pathway. The results
imply that AKT and ERK signaling by CXCR7 may reciprocally regulate each other
in bladder cancer cells, for which it is unclear whether the induced cellular signaling
networks involve in CXCR4-CXCR7 heterodimer complex; however, further studies
are under investigation.
EMT has been noted as a critical process that will contribute to tumor invasive
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phenotypes, associated with multiple activated pathways, including AKT and ERK
signaling et al.[41-43]. Our results show that activating AKT and ERK pathways by
CXCR7 are responsible for in vitro and in vivo EMT process of bladder cancer.
Simultaneously targeting the two pathways by using U0126 and LY294002 inhibitors
or using CCX733, a selective CXCR7 antagonist drastically reduced CXCR7-induced
EMT process. These findings are consistent with the results reported by Burns et
al.[16], in which small molecular antagonists to CXCR7 impeded tumor growth in
vivo. Moreover, Boudot et al. indicated that overexpression of CXCR7 in breast
cancer cell lines increases cell basal proliferation and leads to an estrogen-
independent growth of cells whereby it may play a role in triggering initial step of
EMT [44]. Thus, CXCR7 signaling may be an attractive new therapeutic target for
treatment of multiple tumors, including bladder cancer.
In summary, these data demonstrate a role for CXCR7 in bladder cancer progression.
Elucidation of newly identified receptor roles in tumorigenesis and progression may
indicate new avenues of potentially therapeutic intervention in bladder cancer.
Acknowledgements
We are indebted to Dr. Mark Penfold (ChemoCentryx, USA) for CXCR7 antagonists.
Research in the authors’ laboratory is supported by the National Natural funding of
China (81071747), National key program (973) for Basic Research of China
(2011CB510106,2011CB504300), Shanghai Education Committee Key Discipline
and Specialties Foundation (J50208), the Program for Professor of Special
Appointment (Eastern Scholar to J, Wang) at Shanghai Institutions of Higher
Learning, Shanghai Leading Academic Discipline Project (No. S30201 to Chen Xu)
and Shanghai Pujiang Program (10PJ1406400), Science and
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Technology Development Fund of Shanghai Municipal Health Bureau (2011Y158)
and Ph.D innovation fund from Shanghai Jiao Tong University School of Medicine
(BXJ201103).
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Figure Legends
Figure. 1. CXCR7 expression is correlated with bladder cancer development.
(A) Expression of CXCR7 in human bladder normal (a) and cancer tissues (b, c d).
Formalin-fixed paraffin-embedded tissues were incubated overnight at room
temperature with anti-human CXCR7 antibody. MBA171 (IgG2a) was used as a
negative control. Representative micrographs were taken at an original magnification
20, where the black bars represent 100 µM.
(B) Quantitative histological evaluation of CXCR7 expression. CXCR7 expression
intensity was scored by a genitourinary pathologist on a four point scale as negative
(1), weak (2), moderate (3), or strong (4). Mean expression scores multiplied by
percent positive cells in the field (Quick’s combined score system) are presented for
normal, grade I, grade II and grade III cases in a graphic format using error bars with
95% confidence intervals (CI). * presents statistically significant differences between
normal and cancer, p < 0.001, and # indicates marked differences between grade I
and grade II or grade III, p < 0.001.
Figure. 2. CXCR7 modulates phenotype of bladder cancer cells.
(A) Top: mRNA level of CXCR7 in human bladder cancer cells was detected by RT-
PCR. Bottom: Cell surface expression level of CXCR7 was evaluated by FACS
analysis using mouse anti-human CXCR7 antibody, and mouse anti-human IgG
served as isotype control.
(B) FACS analysis of cell surface CXCR7 expression in lentivirus-transfected bladder
cancer cell lines. A mouse anti-human CXCR7 antibody was used to detect the
overexpressing CXCR7 (J82control/J82CXCR7 and T24control/T24CXCR7)) or in which
CXCR7 expression is reduced using shRNA (J82 shcontrol /J82 shCXCR7 and
RT4shcontrol/RT4shCXCR7). A GFP sequence was incorporated into the shRNA as a
vector control. Mouse anti-human IgG served as isotype control.
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(C) Proliferation of bladder cancer cell lines in response to altered CXCR7 levels.
After a 24-h serum withdrawal, bladder cancer cells were digested and washed three
times in PBS, and 1104 cells were plated in into 96-well flat-bottomed tissue culture
plates in 0.1 ml in complete growth medium. Proliferation was evaluated by XTT
assay over a 4-day period. * denotes significant difference from controls (p < 0.05,
ANOVA) for means S.E. of n = 5 samples per condition.
(D) Restoring expression of CXCR7 is responsible for cell migration. Serum was
withdrawn before analysis to avoid effect of cell proliferation. The migration status
was assessed by measuring the movement of cells into a scraped area created by a 10
l pipette tube, and the spread of wound closure was observed at indicated times after
scratching the surface of a confluent layer of cells. Scale bar: 100 μm. This experience
was performed three times.
(E) CXCR7 regulates bladder cancer cell invasion. Bladder cancer cells were placed
in the top chamber of invasion plates containing a reconstituted extracellular matrix in
serum-free medium, and complete medium and CXCL12 (200 ng/ml) were added to
the lower chambers. Invasion was determined at 48 h by MTT staining and the data
were read on a multiwell scanning spectrophotometer (Thermo) at A580 and
presented as % invasion binding ± standard deviation for n = 5. * denotes significant
difference from controls (p < 0.05, ANOVA).
Figure. 3. CXCR7 expression regulates VEGF and IL-8 secretion.
(A) Detection of cytokines for angiogenesis in array. Detection of cytokines from CM
derived from J82, andT24 cells overexpressing CXCR7 and their respective control
cells. CM from cells was collected after culturing for 24 h. The signals were
visualized by chemolueminescense. Normalization was done by positive control (Pos)
according to the manufacturer protocol. The data indicates common alterations in
VEGF and IL-8 levels, following induced CXCR7 expression.
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(B) CXCR7 regulates secretion of VEGF and IL-8. VEGF and IL-8 levels were
evaluated by ELISA for bladder cancer cells with altered expression of CXCR7 at 48
h. The data are presented as mean ± std. dev. for triplicate determinations and
normalized against total protein. *Denotes significant difference from respective
controls (p < 0.05, ANOVA). n = 6 in groups
(C) Left, effect of bladder cancer expression of CXCR7 on blood vessel formation.
HDMECs were plated in growth factor reduced MatrigelTM in the presence or absence
of CM from bladder cancer cells as a functional in vitro assay of blood vessel
formation. CM from cells was collected after culturing for 24 h. The cultures were
then fixed and stained. As a negative control, only HDMECs were plated. Vessel
sprout formation was quantified by direct microscopic counting. Original
magnification 20 , where the black bars represent 100 µM. Right, the data are
presented as mean ± std. dev. for triplicate determinations. * indicates significant
difference from controls (p < 0.05, ANOVA).
Figure. 4. CXCR7 is involved in regulating the expression of EMT markers.
(A) Western blots were used to assay expression of epithelial marker (E-cadherin) and
mesenchymal markers (N-cadherin and -catenin). Whole cell lysates were
immunoblotted with antibody to E-cadherin, N-cadherin and -catenin. The blots
were stripped and reprobed with GAPDH antibody to confirm equal protein loading.
(B) Potential target gene identification by cDNA microarray analysis. Clustering map
of differentially expressed genes overlapped with cancer-associated genes in CXCR7
shRNA-targeted RT4 cells compared with the average expression of the control. Row
represents gene, column shows experimental cells. Down-regulated genes were
shown in red and up-regulated in green. Detailed data were set in Supplemental
Table1.
(C) Selective fold-change map associated with EMT process in bladder cancer cells.
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Up-regulated genes are listed on the right; and down-regulated genes are listed on the
left.
Figure. 5. CXCR7 mediates activation of AKT, ERK, and STA3 pathways.
(A) Evaluation of the effect of CXCR7 on AKT , ERK and STAT3 signaling. After a
24 h serum withdrawal, whole cell proteins were extracted and lysates were separated
on SDS-PAGE and immunoblotted with antibodies to p-AKT (Thr-308), p-ERK and
p-STAT3 (Tyr-705). The blots were stripped and reblotted with antibodies against
total AKT, ERK and STAT3.
(B) Expression of N-cadherin and -catenin upon inhibiting activation of AKT and
ERK signaling. Cell extracts were prepared from J82CXCR7 cells with the pretreatment
of DMSO (10 μl) or PI3 Kinase Inhibitor LY294002 (5 μM) or MEK1/2 Inhibitor
U0126 (10 μM) or two inhibitors combined for 24 h, and were used to detect the
expressions of total ERK, p-ERK, total AKT, p-AKT (T-308), N-cadherin, -catenin
and GAPDH by Western blot assays.
(C) Expression profile of N-cadherin and -catenin after block by CCX733 and
CCX771, selective CXCR7 antagonists. Cell extracts were prepared from J82CXCR7
cells with the pretreatment of DMSO (0.5 μl) or negtive control CCX704 (0.5 μM) or
CXCR7 inhibitor CCX771 (0.5 μM) and CCX733 (0.5 μM) for 24 h, and were then
used to detect the expressions of N-cadherin, -catenin and GAPDH by Western blot
assays.
Figure. 6. CXCR7 promotes tumor growth, angiogenesis and EMT in vivo.
(A) Effect of CXCR7 on J82 cell growth in vivo. 8-week-old SCID mice were
implanted subcutaneously with 8 105 J82 cells expressing various levels of CXCR7.
Tumor volume (mm3) was evaluated at indicated times. * indicates significant
difference from the controls (p < 0.05).
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(B) Effect of CXCR7 on tumor weight. Representative macroscopic appearance of
tumor growth in J82control (bottom) and J82CXCR7 (top). Tumor weight was measured at
sacrifice. Arrows indicate that abundant blood vessels are observed in tumors bearing
J82CXCR7 cells. The data are presented as mean ± S.D. for triplicate determinations. N
= 8 in groups. Representative 4 tumors are shown. * indicates significant difference
from the controls (p < 0.05).
(C) Histologic evaluation of microvessel growth, and expression of EMT markers in
tumors with altered CXCR7 levels. Subcutaneous tumors expressing various levels of
CXCR7 were harvested and processed for immunohistochemistry staining for p-ERK,
p-AKT (Thr308) , N-cadherin, β-catenin, and human CD31 (1:100 anti-rabbit IgG).
The arrows indicate positive areas. Original magnification 20 , where the black bars
represent 100 µM.
(D) Quantification of microvessel formation in tumors with altered CXCR7 levels.
The numbers of stained microvessels were blindly evaluated in 10 random fields per
implant at 200 magnifications. * indicates significant difference from the controls (p <
0.05,
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Normal
Grade II
A.
a
c
Grade I
b
Grade III
d
Hao et al. Figure 1
* B.
95
% C
I S
tain
ing
In
ten
sity
0
1
2
3
4
5
N=20
N=6
N=33 N=19
*
#
# *
*
Figure1
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Hao et al. Figure 2
0
0.05
0.1
0.15
0.2
0.25
1 2 3 4 5 6
XT
T(O
D4
50
-69
0)
系列1 系列2 系列3 系列4
C.
1 d 2 d 3 d 4 d *
*
*
CXCR7
GAPDH
RT4 J82 T24 A.
IgG
CXCR7
RT4
Co
un
ts
CXCR7-PE 10
0 10
1 10
2 10
3 10
4 0
300
Co
un
ts IgG
CXCR7
J82
10 0
10 1
10 2
10 3
10 4 0
300
CXCR7-PE
IgG
CXCR7
T24
10
0 10
1 10
2 10
3 10
4 0
350
CXCR7-PE
Co
un
ts
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
MT
T(O
D5
90
)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
* *
E.
CXCL12 200 ng/ml
MT
T(O
D5
90
)
B.
10 0
10 1
10 2
10 3
10 4 0
400 RT4shcontrol
RT4shCXCR7
IgG
CXCR7-PE
Co
un
ts
10 0
10 1
10 2
10 3
10 4 0
350
IgG
T24control
T24CXCR7
CXCR7-PE
Co
un
ts
10 0 10 1 10 2 10 3 10 4 0
350
CXCR7-PE
J82shcontrol
J82shCXCR7
IgG
J82CXCR7
Cou
nts
J82control
Figure2
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0
5
10
15
20
25
30
35
40
45*
*
*
J82Control CM
EC
J82CXCR7 CM
RT4shControl CM RT4shCXCR7 CM
T24Control CM T24CXCR7 CM
C.
*
IL-8
(p
g/m
l)
0
200
400
600
800
1000
1200
1400
1600
1800
1 2 3 4 5 6
* *
0
500
1000
1500
2000
2500
3000
3500
4000
1 2 3 4 5 6
VE
GF
(p
g/m
l)
*
*
* B.
T24control T24CXCR7
J82control J82CXCR7
1
1
2
2
1. IL-8
2. VEGF
A.
3
3
3. Pos
Hao et al. Figure 3
Figure3
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Hao et al. Figure 4
-4 -2 0 2 4 6 8
FGFBP1
RPL13A
SERPINE1
SNAI2
MST1R
TGFBI
CDH3
CXCR7
IGFBP6
Up-
regulated
Down-
regulated
Changed fold C.
A.
E-Cadherin
Beta-catenin
N-Cadherin
GAPDH
B.
Figure4
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Hao et al. Figure 5
Akt
GAPDH
CCX704 CCX771 CCX733 DMSO
p-ERK
ERK
C.
p-Akt(308)
-catenin
N-cadherin
DMSO
LY294002 - - +
U0126
+
- - + +
B.
-
-
-catenin
p-Akt (308)
p-Akt (473)
p-ERK
N-Cadherin
ERK
Akt
GAPDH
A.
p-Erk1/2
p-STAT3
p-AKT(T308)
STAT3
AKT
Erk1/2
Figure5
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0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
J82control J82CXCR7
*
Tu
mor
Wei
gh
t (m
g)
J82CXCR7
B.
0
100
200
300
400
500
600
700
800
1 2 3 4
Tu
mor
Volu
me
(mm
)3
20 d 26 d 32 d 38 d
Days Post Implant
A.
J82CXCR7
*
-catenin
J82CXCR7
CD31
C.
N-cadherin
p-Akt (308)
p-ERK
0
2
4
6
8
10
1 2
Sta
ined
Ves
sels
/ F
ield
CD31 Staining
*
D.
Hao et al. Figure 6
Figure6
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0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
J82control J82CXCR7
*
Tu
mor
Wei
gh
t (m
g)
J82CXCR7
CXCR7 promotes bladder cancer growth, angiogenesis and EMT in vivo.
Hao MG, Zheng JH, Hou KL, Wang JL, Chen XS, Lu XJ, Bo JJ, Xue X, Shen KW,Wang JH.
*Graphical Abstract (for review)