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

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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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:jianhuaw2007@gmail.com.

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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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References

[1] Taylor JA 3rd, Kuchel GA. Bladder cancer in the elderly: clinical outcomes, basic

mechanisms, and future research direction. Nat Clin Pract Urol 2009;6:135-44.

[2] Wang Y, Zhong W. Epidemiologic analysis of risk factors of current bladder cancer.

J Contemp Urol Reprod Oncol 2010;2:363-7.

[3] Chromecki TF, Bensalah K, Remzi M, Verhoest G, Cha EK, Scherr DS, et al.

Prognostic factors for upper urinary tract urothelial carcinoma. Nat Rev Urol

2011;8:440-7.

[4] Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4:540-50.

[5] Maksym RB, Tarnowski M, Grymula K, Tarnowska J, Wysoczynski M, Liu R, et al.

The role of stromal-derived factor-1--CXCR7 axis in development and cancer. Eur J

Pharmacol 2009;625:31-40.

[6] Sun X, Cheng G, Hao M, Zheng J, Zhou X, Zhang J, et al. CXCL12 / CXCR4 /

CXCR7 chemokine axis and cancer progression. Cancer Metastasis Rev

2010;29:709-22.

[7] Vindrieux D, Escobar P, Lazennec G. Emerging roles of chemokines in prostate

cancer. Endocr Relat Cancer 2009;16:663-73.

[8] Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the

chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature

1998;393:595-9.

[9] Wang J, Sun Y, Song W, Nor JE, Wang CY, Taichman RS. Diverse signaling

pathways through the SDF-1/CXCR4 chemokine axis in prostate cancer cell lines

leads to altered patterns of cytokine secretion and angiogenesis. Cell Signal

2005;17:1578-92.

[10] Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, Moepps B, et al. The

chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1

in T lymphocytes. J Biol Chem 2005;280:35760-6.

[11] Libert F, Parmentier M, Lefort A, Dumont JE, Vassart G. Complete nucleotide

sequence of a putative G protein coupled receptor: RDC1. Nucleic Acids Res

1990;18:1917.

[12] Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB. The G-protein-coupled

receptors in the human genome form five main families. Phylogenetic analysis,

paralogon groups, and fingerprints. Mol Pharmacol 2003;63:1256-72.

[13] Shimizu N, Soda Y, Kanbe K, Liu HY, Mukai R, Kitamura T, et al. A putative G

protein-coupled receptor, RDC1, is a novel coreceptor for human and simian

Page 22 of 36

Accep

ted

Man

uscr

ipt

22

immunodeficiency viruses. J Virol 2000;74:619-26.

[14] Raggo C, Ruhl R, McAllister S, Koon H, Dezube BJ, Fruh K, et al. Novel cellular

genes essential for transformation of endothelial cells by Kaposi's sarcoma-associated

herpesvirus. Cancer research 2005;65:5084-95.

[15] Martinez A, Kapas S, Miller MJ, Ward Y, Cuttitta F. Coexpression of receptors for

adrenomedullin, calcitonin gene-related peptide, and amylin in pancreatic beta-cells.

Endocrinology 2000;141:406-11.

[16] Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, et al. A novel

chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion,

and tumor development. J Exp Med 2006;203:2201-13.

[17] Tripathi V, Verma R, Dinda A, Malhotra N, Kaur J, Luthra K. Differential expression

of RDC1/CXCR7 in the human placenta. J Clin Immunol 2009;29:379-86.

[18] Miao Z, Luker KE, Summers BC, Berahovich R, Bhojani MS, Rehemtulla A, et al.

CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on

tumor-associated vasculature. Proc Natl Acad Sci U S A 2007;104:15735-40.

[19] Wang J, Shiozawa Y, Wang Y, Jung Y, Pienta KJ, Mehra R, et al. The role of

CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. J Biol

Chem 2008;283:4283-94.

[20] Rajagopal S, Kim J, Ahn S, Craig S, Lam CM, Gerard NP, et al. Beta-arrestin- but

not G protein-mediated signaling by the "decoy" receptor CXCR7. Proc Natl Acad

Sci U S A 2010;107:628-32.

[21] Kalatskaya I, Berchiche YA, Gravel S, Limberg BJ, Rosenbaum JS, Heveker N.

AMD3100 is a CXCR7 ligand with allosteric agonist properties. Mol Pharmacol

2009;75:1240-7.

[22] Luker KE, Gupta M, Steele JM, Foerster BR, Luker GD. Imaging ligand-dependent

activation of CXCR7. Neoplasia 2009;11:1022-35.

[23] Zabel BA, Wang Y, Lewen S, Berahovich RD, Penfold ME, Zhang P, et al.

Elucidation of CXCR7-mediated signaling events and inhibition of CXCR4-mediated

tumor cell transendothelial migration by CXCR7 ligands. J Immunol 2009;183:3204-

11.

[24] Marechal R, Demetter P, Nagy N, Berton A, Decaestecker C, Polus M, et al. High

expression of CXCR4 may predict poor survival in resected pancreatic

adenocarcinoma. British journal of cancer 2009;100:1444-51.

[25] Liang Z, Yoon Y, Votaw J, Goodman MM, Williams L, Shim H. Silencing of

CXCR4 blocks breast cancer metastasis. Cancer research 2005;65:967-71.

[26] Hou K, Zhang L, Bo J, Hao M, Yang G, Jiang H, et al. Expression of CXCR7 protein

in human bladder cancer and its clinical significance. Chinese Journal of Urology

2011;32:42-6.

Page 23 of 36

Accep

ted

Man

uscr

ipt

23

[27] Zeisberg M, Neilson EG. Biomarkers for epithelial-mesenchymal transitions. The

Journal of clinical investigation 2009;119:1429-37.

[28] Madden SL, Cook BP, Nacht M, Weber WD, Callahan MR, Jiang Y, et al. Vascular

gene expression in nonneoplastic and malignant brain. Am J Pathol 2004;165:601-8.

[29] Shimizu S, Brown M, Sengupta R, Penfold ME, Meucci O. CXCR7 protein

expression in human adult brain and differentiated neurons. PloS one 2011;6:e20680.

[30] Hou KL, Hao MG, Bo JJ, Wang JH. CXCR7 in tumorigenesis and progression.

Chinese journal of cancer 2010;29:456-9.

[31] Fidler IJ. Selection of successive tumour lines for metastasis. Nature: New biology

1973;242:148-9.

[32] Fureder W, Krauth MT, Sperr WR, Sonneck K, Simonitsch-Klupp I, Mullauer L, et al.

Evaluation of angiogenesis and vascular endothelial growth factor expression in the

bone marrow of patients with aplastic anemia. Am J Pathol 2006;168:123-30.

[33] Jubb AM, Hurwitz HI, Bai W, Holmgren EB, Tobin P, Guerrero AS, et al. Impact of

vascular endothelial growth factor-A expression, thrombospondin-2 expression, and

microvessel density on the treatment effect of bevacizumab in metastatic colorectal

cancer. J Clin Oncol 2006;24:217-27.

[34] Singh RK, Lokeshwar BL. The IL-8-regulated chemokine receptor CXCR7

stimulates EGFR signaling to promote prostate cancer growth. Cancer research

2011;71:3268-77.

[35] Grymula K, Tarnowski M, Wysoczynski M, Drukala J, Barr FG, Ratajczak J, et al.

Overlapping and distinct role of CXCR7-SDF-1/ITAC and CXCR4-SDF-1 axes in

regulating metastatic behavior of human rhabdomyosarcomas. International journal of

cancer 2010;127:2554-68.

[36] Odemis V, Boosmann K, Heinen A, Kury P, Engele J. CXCR7 is an active

component of SDF-1 signalling in astrocytes and Schwann cells. Journal of cell

science 2010;123:1081-8.

[37] Hattermann K, Held-Feindt J, Lucius R, Muerkoster SS, Penfold ME, Schall TJ, et al.

The chemokine receptor CXCR7 is highly expressed in human glioma cells and

mediates antiapoptotic effects. Cancer research 2010;70:3299-308.

[38] Levoye A, Balabanian K, Baleux F, Bachelerie F, Lagane B. CXCR7 heterodimerizes

with CXCR4 and regulates CXCL12-mediated G protein signaling. Blood

2009;113:6085-93.

[39] Hartmann TN, Grabovsky V, Pasvolsky R, Shulman Z, Buss EC, Spiegel A, et al. A

crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12-

triggered integrin activation but not in chemokine-triggered motility of human T

lymphocytes and CD34+ cells. J Leukoc Biol 2008;84:1130-40.

[40] Decaillot FM, Kazmi MA, Lin Y, Ray-Saha S, Sakmar TP, Sachdev P.

Page 24 of 36

Accep

ted

Man

uscr

ipt

24

CXCR7/CXCR4 heterodimer constitutively recruits {beta}-arrestin to enhance cell

migration. J Biol Chem 2011;286:32188-97.

[41] Godoy P, Hengstler JG, Ilkavets I, Meyer C, Bachmann A, Muller A, et al.

Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid

dedifferentiation and transforming growth factor beta-induced apoptosis. Hepatology

2009;49:2031-43.

[42] Irie HY, Pearline RV, Grueneberg D, Hsia M, Ravichandran P, Kothari N, et al.

Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial-

mesenchymal transition. J Cell Biol 2005;171:1023-34.

[43] Tommasi S, Pinto R, Pilato B, Paradiso A. Molecular pathways and related target

therapies in liver carcinoma. Curr Pharm Des 2007;13:3279-87.

[44] Boudot A, Kerdivel G, Habauzit D, Eeckhoute J, Le Dily F, Flouriot G, et al.

Differential estrogen-regulation of CXCL12 chemokine receptors, CXCR4 and

CXCR7, contributes to the growth effect of estrogens in breast cancer cells. PloS one

2011;6:e20898.

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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

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)

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

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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

*

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g)

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0

100

200

300

400

500

600

700

800

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me

(mm

)3

20 d 26 d 32 d 38 d

Days Post Implant

A.

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CD31

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0

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6

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*

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Hao et al. Figure 6

Figure6

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0

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0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

J82control J82CXCR7

*

Tu

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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)