Breast Cancer Research and Treatment

, Volume 131, Issue 3, pp 837–848

The chemokine receptor CCR4 promotes tumor growth and lung metastasis in breast cancer

Authors

  • Ji-Yu Li
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
  • Zhou-Luo Ou
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
  • San-Jian Yu
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
  • Xiao-Li Gu
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
  • Chen Yang
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
  • Ao-Xiang Chen
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
  • Gen-Hong Di
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
  • Zhen-Zhou Shen
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
    • Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College, Institutes of Biomedical ScienceFudan University
    • Department of Breast Surgery, Cancer Hospital/Cancer Institute, Breast Cancer InstituteFudan University
Preclinical study

DOI: 10.1007/s10549-011-1502-6

Cite this article as:
Li, J., Ou, Z., Yu, S. et al. Breast Cancer Res Treat (2012) 131: 837. doi:10.1007/s10549-011-1502-6

Abstract

Increasing evidence has shown that chemokines and chemokine receptors are associated with tumor growth and metastasis. CCR4, an important chemokine receptor for regulating immune homeostasis, is thought to be involved in hematologic malignancies and has also recently implicated in some solid tumors, such as gastric cancer. The possible role of CCR4 in breast cancer has not been well elucidated. In this study, we show that CCR4 is differentially expressed in human breast cancer cell lines. Specifically, we find that CCR4 is overexpressed in breast cancer cell lines with high metastatic potential. More importantly, we used a combination of overexpression and RNA interference to demonstrate that CCR4 promotes breast tumor growth and lung metastasis in mice. Furthermore, we find that microvessel density is significantly increased in tumors formed by CCR4-overexpressing cells and decreased in those formed by CCR4-knockdown cells. We find that overexpression of CCR4 can enhance the chemotactic response of breast cancer cells to CCL17. However, the expression of CCR4 does not affect the proliferation of breast cancer cells in vitro. Furthermore, we show that CCR4 expression is positively correlated with HER2 expression, tumor recurrence and lymph node, lung and bone metastasis (P < 0.05). Multivariate analysis showed that CCR4 expression is a significant independent prognostic factor for overall survival (P = 0.036) but not for disease-free survival in patients with breast cancer (P = 0.071). Survival analysis indicated a strong association between CCR4 expression and lower overall survival (P = 0.0001) and disease-free survival (P = 0.016) in breast cancer.

Keywords

Breast cancerMetastasisCCR4Chemokine

Introduction

Breast cancer is one of the most common malignancies in women worldwide [1]. The molecular basis of the complex biochemical processes involved in breast cancer, including tumor growth and metastasis, is becoming increasingly understood. Mounting evidence has shown that chemokines and chemokine receptors are involved in tumor cell proliferation, survival, migration, and angiogenesis as well as metastasis [2, 3]. Chemokines belong to a superfamily of small molecules (8–14 kDa) that were initially characterized because their interaction with chemokine receptors was associated with promoting leukocyte infiltration to inflammatory sites [4]. Cancer cells can also express chemokine receptors and respond to specific chemokines. Muller et al. proposed that tumor cells use chemokine-mediated mechanisms, such as those regulating leukocyte trafficking, during the process of metastasis. They demonstrated that the chemokine CXCL12 and its receptor CXCR4 have critical roles in the process of organ-specific metastasis in breast cancer, and the expression of CCR7 in breast cancer correlates with lymph node metastasis and poor prognosis [5]. Increasing numbers of chemokines and chemokine receptors are thought to be involved in breast cancer. CCR5 is involved in breast cancer progression in a p53-dependent manner [6]. CCR5 blockade significantly enhances the proliferation of breast cancer xenografts bearing wild-type p53. A recent study by Miao et al. [7] revealed that another CXCL12 receptor, CXCR7, could promote breast cancer tumor growth and metastasis in vivo. Our previous studies have shown that chemokine decoy receptors, including DARC, D6 and CCX-CKR, play negative roles in tumorigenesis and metastasis in human breast cancer and are related to the prognosis of patients with breast cancer [811]. All these data indicate that clearly elucidating the function of chemokines and chemokine receptors in breast cancer is crucial for understanding of the biological behavior of breast cancer.

CCR4 is an important chemokine receptor for regulating immune homeostasis and is selectively expressed on Th2 cells and regulatory T cells [12]. And CCR4 plays a critical role in hematologic malignancies including adult T-cell leukemia/lymphoma, peripheral T cell lymphoma, acute myeloid lymphoblastic leukemia and chronic lymphocytic leukemia [1315]. Targeting CCR4 has become a novel strategy in the treatment of these diseases [16]. Lee et al. [17] recently published the first report of a role for CCR4 in solid tumors. They showed that CCR4 is expressed in 17.0% primary gastric cancers and is associated with a poor prognosis in patients with gastric cancer. In the metastatic 4T1 mouse mammary carcinoma model, Olkhanud et al. [18] found that only CCR4-positive tumor cells had the ability to metastasize to the lung, and that strategies that target CCR4 could efficiently control lung metastasis.

Although the function of CCR4 in breast cancer has been partially revealed using a mouse mammary carcinoma model, little is known about the function of CCR4 in human breast cancer. In the present study, the possible role of CCR4 in the progression and metastasis of human breast cancer was investigated. The correlation between CCR4 expression and prognosis in patients with breast cancer was also assessed.

Methods and materials

Cell lines

Human breast cancer cell lines MDA-MB-231, MCF-7, BT-474, SK-BR-3, MDA-MB-468, and T47D were purchased from the American Type Culture Collection. The highly metastatic MDA-MB-231HM and MDA-MB-231-B cell line were established by our institute. The MDA-MB-231HM cell line has a high potential to metastasize to the lung and its establishment has been described previously [19]. The MDA-MB-231-B cell line was obtained from bone metastases resulting from MDA-MB-231.

Western blot

Western blot using goat anti-human CCR4 antibody (ab1669, Abcam) was done according standard protocols. Chemiluminescent detection were performed and images were captured by the FUJIFILM LAS-3000 system (Fujifilm, Tokyo, Japan).

Flow cytometric analysis

Cells were stained with PE-labeled mouse anti-human CCR4 (551120, BD Pharmingen™). Samples were analyzed using a FACSCalibur system (Becton–Dickinson Biosciences, San Diego, CA) [20] with ModFit LT™ software (Verity Software House, Inc., Topsham, ME).

Stable overexpression of CCR4

The coding sequence of CCR4 (1083 bp) was kindly provided by Professor Ying Wang. We cloned CCR4 into the pCDH-CMV-MCS-Puro expression vector (System Biosciences, Mountain View, CA, USA). Constructs were confirmed by sequencing. The pCDHpPACKH1™ Lentivector Packaging Kit (System Biosciences, Mountain View, CA, USA) was used to produce lentivirus particles. MDA-MB-231 cells were infected with the lentivirus particles according to the manufacturer’s protocol. The empty vector was packaged as a negative control. Stable transfectants were selected and cultured in medium containing 3 ng/μl puromycin.

Stable RNA interference against CCR4

We designed short hairpin RNA molecules targeted against sites beginning at nucleotides 348 and 926 of human CCR4. The following oligonucleotides were synthesized (Sangon, Shanghai) (1) position 348, 5′-ccggGGTTCTGGTCCTGTTCAAATActcgagTATTTGAACAGGACCAGAACCtttttg-3′ and 5′-aattcaaaaaGGTTCTGGTCCTGTTCAAATActcgagTATTTGAACAGGACCAGACC-3′ and (2) position 926 5′-ccggGGTTCTGGACACCTTACAACActcgagTGTTGTAAGGTGTCCAGAACCtttttg-3′ and 5′-aattcaaaaaGGTTCTGGACACCTTACAACActcgagTGTTGTAAGGTGTCCAGAACC-3′ (lowercase letters represent linkers).

Oligonucleotides were annealed and inserted into digested PLKO.1-puro. Production of the lentiviral particles were carried out according to the manufacturer’s protocol. MDA-MB-231HM cells were infected with lentivirus particles containing the shRNA and stable transfectants were selected and cultured in medium containing 2 ng/μl puromycin. The empty PLKO.1 puro plasmid was packaged as a negative control. The PLKO.1 puro plasmid, packaging plasmid, pCMV-dR8.91 and envelope VSV-G were purchased from Addgen (Cambridge, MA, USA).

Migration assay

A migration assay was conducted with a 24-well cell culture chamber using inserts with 8-μm sized pores (Corning Costar). The breast cancer cells were suspended in chemotaxis buffer (DMEM with 0.1% BSA and 12 mM HEPES) at 2 × 105/ml and 0.5 ml of the suspension was added to the inserts. Buffer either with or without CCL17 (10 ng/ml 364-DN/CF, R&D System) was added into the lower chamber. After a 24-h incubation at 37°C, the cells on the lower surface of the membrane were stained with crystal violet and counted by microscopy in 5 different fields (×200).

Proliferation assay

Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies Inc., Gaithersburg, MD) assays. Cells well were plated in 96-well plates at 1 × 104/well in media either with or without CCL17 (100 ng/ml), which was changed everyday. To evaluate proliferation, 10 μl of CCK-8 was added per well, and the cells were incubated for an additional 4 h, after which the absorbance at 450 nm was recorded using a 96-well plate reader (Sunrise Microplate Reader, Tecan US, Inc.,Charlotte, NC).

Animal experiments

The animals used in this study were 4–6-week-old athymic female BALB/c nu/nu mice, which were provided by Shanghai Institute of Materia Medica, Chinese Academy of Science. Breast tumor cells were implanted into the mammary fat pad as described previously [21]. Cells (106) were inoculated into the anesthetized mice in 100 μl of culture medium. Forty-two nude mice were divided into seven groups in this study, and each group had six mice. The animals were monitored every 2 days, for up to 5 weeks, for tumor growth and general health. The rate of primary tumor growth was determined by plotting the means of two orthogonal diameters of the tumors, measured at 7-day intervals. The tumor volumes were calculated using the following formula: volume = 0.52 × width2 × length. The animals were killed and autopsied 5 weeks after tumor inoculation. Metastasis formation was assessed by macroscopic observation of all the major organs for secondary tumors and was confirmed by histological examination of the organs. The lung tissues were serially cut into 5-mm slices, and every 10th section was stained with H&E to evaluate the presence or absence of lung metastasis. Two independent pathologists calculated the number of metastases in whole lungs.

Immunohistochemistry

Tumor sections were subjected to immunohistochemical staining for CCR4 and CD34. Tumor sections were incubated in a 1:200 dilution of goat anti-human CCR4 (ab1669, Abcam) and a 1:50 dilution of rabbit anti-mouse CD34 (ab8158, ABcam). Primary antibodies were detected with HRP-conjugated secondary antibodies followed by colorimetric detection with 3,3-diaminobenzidine (DAB). Paraffin blocks of 483 patient tumor samples were reviewed for immunohistochemistry. Informed consent forms were obtained from all the patients. This study was approved in advance by the Institutional Review Board of the Cancer Hospital, Fudan University.

The tumor sections were evaluated microscopically by two independent investigators, who were blinded to the patient outcomes. The results of CCR4 immunostaining were interpreted as positive if 10% or more of the tumor cells showed cytoplasmic or membrane staining.

Enzyme linked immunosorbent assay analysis

The levels of mouse CCL17 and CCL22 in xenografts were determined using an enzyme linked immunosorbent assay (ELISA) kit (Mouse CCL17/TARC DuoSet and Mouse CCL22/MDC DuoSet, R&D Systems Inc., USA). The CCL17 and CCL22 concentrations in the supernatants of homogenized xenografts were determined in duplicate, according to the instruction booklets supplied by the manufacturers.

Statistical analysis

Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software Version 11.5 for Windows (SPSS Inc., Chicago, IL). ANOVA and Student’s t tests were used to determine the statistical significance of differences between experimental groups. Values of P < 0.05 were considered statistically significant. Graphs were created with GraphPad Prism 5.

Results

Expression of CCR4 in breast cancer cell lines

We performed western blots to investigate the mRNA expression levels of CCR4 in 6 human breast cancer cell lines. We found that CCR4 protein was differentially expressed in these breast cancer cell lines and was relatively highly expressed in a HER2 positive cell line, SK-BR-3(Fig. 1a). Flow cytometric analysis confirmed these findings. The frequency of CCR4-positive cells is shown in Fig. 1b.
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Fig. 1

Expression of CCR4 in breast cancer cell lines. Expression of CCR4 in 6 human cancer breast cell lines by Western blot (a) and Flow cytometric analysis (b). For a and b: lane 1 MDA-MB-231, lane 2 MCF-7, lane 3 BT-474, lane 4 SK-BR-3, lane 5 MDA-MB-468, lane 6 T47D. Expression of CCR4 in MDA-MB-231 series cells was compared by Western blot (c) and Flow cytometric analysis (d). For c and d: lane 1 MDA-MB-231, lane 2 MDA-MB-231HM, lane 3 MDA-MB-231-B

CCR4 is overexpressed in MDA-MB-231HM and MDA-MB-231-B cells

To examine the role of CCR4 in MDA-MB-231, MDA-MB-231HM and MDA-MB-231-B cells, we investigated the CCR4 expression level in these cells by western blot and flow cytometry. As shown in Fig. 1c, d, CCR4 were overexpressed in both MDA-MB-231HM and MDA-MB-231-B cells compared with their parent cells. These findings suggest a potential correlation between the expression of CCR4 and the metastatic ability of MDA-MB-231 cells.

Lentiviral-mediated CCR4 overexpression in MDA-MB-231 cells and knockdown in MDA-MB-231HM cells

To quantify the direct contribution of CCR4 in breast cancer, we used lentiviral methods to generate MDA-MB-231 cells that stably overexpressed CCR4, which we termed MDA-MB-231/CCR4 cells. An empty expression vector control cell line was also obtained and used to generate cells that we termed MDA-MB-231/vector. CCR4 overexpression was identified by western blot and flow cytometric analysis (Fig. 2a, b).
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Fig. 2

Expression of CCR4 in MDA-MB-231 cells and MDA-MB-231HM cells with gene modification. CCR4 Overexpression in MDA-MB-231 cells was detected by Western blot (a) and Flow cytometric analysis (b). CCR4 knockdown in MDA-MB-231HM cells was detected by Western blot (c) and Flow cytometric analysis (d). For a and b: lane 1 MDA-MB-231, lane 2 MDA-MB-231/vector, lane 3 MDA-MB-231/CCR4. For c and d: lane 1 MDA-MB-231HM, lane 2 MDA-MB-231HM/control, lane 3 231HM/CCR4-RNAi-348, lane 4 231HM/CCR4-RNAi-926

To test the effects of downregulation of CCR4 in breast cancer, we generated MDA-MB-231HM cells harboring stable CCR4 RNA interference expression vectors. Two independent sites were targeted, beginning at nucleotide 348 and 926, to generate 231HM/CCR4-RNAi-348 cells and 231HM/CCR4-RNAi-926 cells, respectively. CCR4 downregulation in these cells verified by western blot and flow cytometric analysis (Fig. 2c, d). MDA-MB-231HM negative control cells were also generated.

CCR4 expression does not affect the proliferation of breast cancer cells in vitro

Proliferation assays were performed to evaluate the potential changes in breast cancer cell proliferation after CCR4 up-regulation or down-regulation. Following stimulation with CCL17, the proliferative potential of MDA-MB-231/CCR4 cells was not significantly increased relative to MDA-MB-231 or MDA-MB-231/vector cells (Fig. 3a). Similar results were observed for the MDA-MB-231HM cells. The proliferation curves of 231HM/CCR4-RNAi-348 and 231HM/CCR4-RNAi-926 cells were similar to the curve of the MDA-MB-231HM cells (Fig. 3b). Furthermore, no changes in proliferation were observed under normal culture conditions without high concentrations of CCL17 (data not shown). In addition, no obvious changes in cell cycle distribution could be seen in the cells following CCR4 overexpression or knockdown (data not shown).
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Fig. 3

CCR4 expression does not affect the proliferation of breast cancer cells in vitro and enhances chemotactic responses of breast cancer cells to CCL17. a The growth curves for MDA-MB-231/CCR4, empty vector and the parents cells in in vivo proliferation assay. b The growth curves for 231HM/CCR4-RNAi-348, 231HM/CCR4-RNAi-926, empty vector and parents cells. Chemotactic responses greatly enhanced in MDA-MB-231/CCR4 cells (c) and decreased in CCR4 knockdown cells (d). *P < 0.05. Columns, mean of three independent experiments; bars, SD. HP, high power objective

Expression of CCR4 enhances chemotactic responses of breast cancer cells to CCL17

A transwell migration assay was performed to test whether the ability of breast cancer cells to migrate in response to CCL17 changes with CCR4 expression levels. As expected, the MDA-MB-231/CCR4 cells exhibited a greatly enhanced chemotactic response compared with either the MDA-MB-231 or MDA-MB-231/vector cells (P < 0.05, Fig. 3c). Similar results were observed for the MDA-MB-231HM cells. The 231HM/CCR4-RNAi-348 and 231HM/CCR4-RNAi-926 cells, in which CCR4 was downregulated, exhibited a significantly decreased chemotactic response to CCL17 (P < 0.05, Fig. 3d). CCR4 levels are closely related with the chemotactic response of breast cancer cells to CCL17.

CCR4 promotes breast tumor growth and lung metastasis in mice

To further investigate whether CCR4 could promote breast tumor growth in vivo, we used an orthotropic xenograft tumor models in nude mice. As Fig. 4a and b shows, MDA-MB-231/CCR4 cells formed larger tumors than either MDA-MB-231 or MDA-MB-231/vector control cells (P < 0.01). 231HM/CCR4-RNAi-348 and 231HM/CCR4-RNAi-926 cells formed smaller tumors than wild-type MDA-MB-231HM cells or negative control MDA-MB-231HM cells (Fig. 4c, d) (P < 0.01). All the primary tumors were evaluated by pathologic examination. The results showed that microvessel density was significantly increased in tumors formed by CCR4-overexpressing cells and decreased in tumors formed by CCR4-knockdown cells (Fig. 5a, c, d, f). These data suggest that CCR4 could enhance tumor growth by promoting blood vessel formation.
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Fig. 4

CCR4 promotes the tumor growth in vivo. a, b Volumes of CCR4 overexpressing tumors were larger than those in wild type tumors and vector control tumors (P < 0.01). c, d Volumes of CCR4 knockdown tumors were significantly smaller than those in wild type tumors and control tumors (P < 0.01). Six mice in each group were used in this study. Points, mean of tumors volume; bars, SD

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Fig. 5

CCR4 promotes lung metastasis in mice. a, d Metastasis to lung (H&E, 100×) and CD34+ staining (100×, as a marker of microvessels) in the final week of control xenografts and CCR4 modified xenografts. b, c, e, f Quantification of average metastasis number of per lung and average number of microvessel density of the control xenografts and CCR4 modified xenografts. *P < 0.05. Columns, mean of three independent experiments; bars, SD

The number of lung metastatic nodules was counted to determine whether the overexpression of CCR4 in breast cancer cells could enhance lung metastasis. MDA-MB-231/CCR4 cells led to more metastatic nodules than either wild type or vector control cells (Fig. 5a, b). Similarly, mice injected with the CCR4-knockdown 231HM/CCR4-RNAi-348 and 231HM/CCR4-RNAi-926 cells exhibited fewer pulmonary metastatic nodules than those injected with either MDA-MB-231HM or control cells (Fig. 5d, e). These data demonstrate that CCR4 expression can enhance the ability of breast cancer cells to metastasize to the lung.

CCL17 and CCL22 levels are related to the CCR4 expression levels in xenograft tumors

Given that CCL17 and CCL22 are closely related to tumor immunity, we examined the concentration of CCL17 and CCL22 in the various of xenograft tumors. We observed significantly increased levels of CCL17 and CCL22 in the tumors formed by CCR4 overexpressing MDA-MB-231/CCR4 cells compared with those formed by either MDA-MB-231 or vector control cells (Fig. 6a). Conversely, CCL17 and CCL22 levels were decreased in 231HM/CCR4-RNAi-348 and 231HM/CCR4-RNAi-926 tumors (Fig. 6b). These results indicated that the concentration of CCL17 and CCL22 in xenograft tumors is related with CCR4 expression.
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Fig. 6

CCL17 and CCL22 levels are related to the CCR4 expression levels in xenograft tumors. a ELISA analysis of CCL17 and CCL22 levels in tumors formed by CCR4 overexpressing MDA-MB-231/CCR4, MDA-MB-231, and vector control cells. b ELISA analysis of CCL17 and CCL22 levels in tumors formed by 231HM/CCR4-RNAi-348, 231HM/CCR4-RNAi-926, MDA-MB-231HM, and control cells. *P < 0.05. Columns, mean of three independent experiments; bars, SD

CCR4 expression is associated with poor prognosis in patients with breast cancer

To determine whether CCR4 expression is associated with clinical and pathologic features in human breast cancer, we performed immunohistochemical staining on samples from 483 patients with primary invasive breast cancer (Fig. 7a). Clinical and pathologic factors are listed in Table 1. Of this group of patients, 100 (20.7%) were CCR4 positive. CCR4 expression was associated with HER2 expression, lymph node metastasis and the frequency of disease recurrence (P < 0.05). CCR4 expression is also correlated with cancer cells metastasis to lung and bone, which are common metastatic sites of breast cancer (P < 0.05).
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Fig. 7

CCR4 expression is associated with poor prognosis in patients with breast cancer. a Representative CCR4 positive human primary breast tumor tissue samples with immunohistochemistry staining. b Kaplan–Meier survival curves indicated a strong association between CCR4 expression and poorer overall survival. c CCR4-positive patients had a lower disease-free survival

Table 1

Clinicopathological characteristics of the patients

 

CCR4 negative (n = 383)

CCR4 positive (n = 100)

P value

Mean age (years)

54.58

53.39

0.346

Tumor size

  

0.246

 T1

157

42

 

 T2

209

52

 

 T3

10

6

 

Not available

7

0

 

Grade

  

0.892

 I

6

1

 

 II

228

61

 

 III

103

26

 

Not available

46

12

 

Lymph node status

  

0.016*

 Positive

205

40

 

 Negative

173

60

 

ER

  

0.343

 Positive

228

54

 

 Negative

146

43

 

 Not available

9

3

 

PR

  

0.650

 Positive

221

55

 

 Negative

152

42

 

 Not available

10

3

 

Her-2

  

0.001*

 Positive

86

39

 

 Negative

296

60

 

 Not available

1

1

 

Recurrence

  

0.013*

 Yes

68

29

 

 No

309

70

 

 Not available

6

1

 

Recurrence site

   

 Local regional

33

9

0.922

 Lung

21

15

0.001*

 Bone

26

13

0.042*

 Liver

7

2

0.910

 Others

9

3

0.710

P < 0.05

The results of multivariate analysis by Cox proportional hazards models showed that, in addition to lymph node and HER2 status, CCR4 expression was also a significant independent prognostic risk factor for overall survival (P = 0.036, Table 2), but not for disease-free survival (P = 0.071, Table 3). Kaplan–Meier survival curves indicated a strong association between CCR4 expression and poorer overall survival (P = 0.0001, Fig. 7b). CCR4- positive patients also had a lower disease-free survival (P = 0.016, Fig. 7c).
Table 2

Multivariate analysis of overall survival by Cox proportional hazards models

Patients

P

Hazard ratio (95% confidence interval)

Age

0.134

Tumor size T1 vs T2 vs T3

0.964

Nuclear grade, I vs II vs III

0.640

Axilla status, positive vs negative

0.030

5.363 (1.182–24.341)

Pathologic types, invasive ductal cancer vs others

0.287

Human epidermal growth factor-2, positive vs negative

0.023

3.322 (1.180–9.349)

CCR4 status, positive vs negative

0.036

2.257 (1.071–7.637)

Table 3

Multivariate analysis of disease-free survival by Cox proportional hazards models

Patients

P

Hazard ratio (95% confidence interval)

Age

0.527

Tumor size T1 vs T2 vs T3

0.174

Nuclear grade, I vs II vs III

0.072

Axilla status, positive vs negative

6.33 × 10−5

2.762 (1.679–4.543)

Pathologic types, invasive ductal cancer vs others

0.870

Human epidermal growth factor-2, positive vs negative

0.003

1.934 (1.253–2.986)

CCR4 status, positive vs negative

0.071

Discussion

Although many molecular mechanisms have been implicated in cancer progression, the critical mechanisms that truly promote breast cancer growth and metastasis are still not completely understood. An increasing number of chemokines and their receptors have been shown to be involved in these complex processes. In the present study, we demonstrated that the chemokine receptor CCR4 could promote tumor growth and lung metastasis in human breast cancer. Furthermore, we found that CCR4 expression was an independent prognostic factor for overall survival and associated with reduced disease-free survival in patients with breast cancer.

Multiple chemokines and their receptors have been shown to be associated with tumor growth [7, 22, 23]. In previous study, we have demonstrated chemokine decoy receptors, including DARC, D6 and CCX-CKR, play a negative role in tumor growth [810]. Here we demonstrate, for the first time, CCR4 expression plays a significant role in tumor growth in murine models of breast cancer. In our study, tumors formed by CCR4-overexpressing MDA-MB-231/CCR4 cells were significantly larger than those formed by MDA-MB-231 cells, whereas tumors formed by CCR4-knockdown cells were significantly smaller. In vitro experiments did not reveal any significant differences between the proliferation of CCR4-overexpressing and wild type cells. These results are consistent with Lee et al.’s [17] findings in gastric cancer cells. Due to the complex tumor microenvironment, a number of factors may play a role in vivo but not in vitro [24]. Questions still remain regarding the mechanism by which CCR4 promotes tumor growth.

Angiogenesis is a complex process that appears to be critical for tumor growth [25]. Previous studies have shown that chemokines are involved in angiogenesis and that the proangiogenic activity of chemokines is mainly mediated through CXCR1 and CXCR2 [26]. We found that microvessel density was increased in xenograft tumors formed by CCR4-overexpressing cells and was decreased in tumors resulting from CCR4-knockdown cells. Lo et al. [27] found that CCR4 and its ligands contributed to the formation of vascular structure by mobilizing smooth muscle precursor cells. These results suggest that CCR4, a chemokine receptor belonged to the CCR family, can promote tumor growth through neovascularization. Further study is needed to determine whether this role can be blocked by anti-angiogenesis agents, such as bevacizumab, and this experiment would be helpful in understanding the mechanism of CCR4 in promoting blood vessel formation. We found that CCL17 and CCL22 levels in xenograft tumors changed with CCR4 expression: they were significantly increased in CCR4-overexpressing tumors and significantly decreased in CCR4-knockdown tumors. It has been reported that CCL17 and CCL22 can recruit more regulatory T cells to help tumor cells escape host immunity [28]. Increasing tumor cells survival can, in turn, lead to larger tumors. It should be noted, however, that CCL17 and CCL22 mRNA expression levels are almost identical in CCR4-overexpressing, CCR4-knockdown and parent cells (data not shown). The mechanism by which CCL17 and CCL22 levels are regulated with CCR4 expression remain unclear and the exact function of CCR4 in promoting tumor growth has yet to be identified. Additional studies are needed to answer these questions.

Olkhanud et al. [18] have described an important role for expression of CCR4 in breast cancer lung metastasis. In their study, they injected mice via the tail vein with 4T1 cell, a murine cancer cell line, and concluded that breast cancer lung metastasis requires CCR4 expression and regulatory T cells. In the present study, we confirm the critical role of CCR4 in breast cancer lung metastasis using MDA-MB-231 cells, a human breast cancer cell line. Instead of injecting mice with cancer cells via tail vein, we injected breast cancer cells into the mammary fat pad of mice which is more consistent with the natural process of breast cancer metastasis. We demonstrated that overexpression of CCR4 can enhance chemotactic response of breast cancer cells to CCL17. A previous study has suggested that primary tumors can induce the production of CCR4 ligands in the lungs of mice [18]. It is therefore conceivable that CCR4 positive tumor cells could migrate to lungs more easily.

We compared the CCR4 expression level of MDA-MB-231HM, MDA-MB-231-B, and MDA-MB-231 cells. We found that CCR4 was overexpressed in MDA-MB-231HM cells, which exhibit a high potential to metastasize to the lung. CCR4 was also overexpressed in MDA-MB-231-B cells, a cell line obtained from bone metastases resulting from MDA-MB-231. Tumor cells that metastasized to the lung and bone were selected for their higher metastatic potential, and the fact that these cells were CCR4-positive indicates that CCR4 is overexpressed in tumor cells with higher metastatic potential.

In clinical samples of patients with invasive breast cancer, 100 of 483 (20.7%) tumors expressed CCR4. Multivariate analysis showed that CCR4 was an independent risk factor for overall survival in breast cancer. Further studies are needed to confirm this new potential prognostic factor in breast cancer. CCR4 expression was also correlated with lung metastasis in patients. This result was consistent with our in vivo and vitro experiments. Bone metastasis is common in breast cancer. In the present study, we observed, for the first time, that CCR4 was related with breast cancer bone metastasis in both in vitro experiments and patients. CCR4 expression was associated with HER2 expression, lymph node metastasis and a high frequency of recurrence, indicating that CCR4 is related to the aggressiveness of breast cancer. In our in vivo experiment, we paid little attention to lymph node metastasis during macroscopic observation, and we did not examine the bone with animal CT or X-ray. Fluorescently labeled cells combined with fluorescence imaging would be useful to determine whether CCR4 expression could affect breast cancer metastasis to lymph nodes or bone in vivo, and further study is needed.

HER2 can affect the expression of chemokine receptors. Li et al. [29] have shown that HER2 could enhance the expression of CXCR4, which was required for HER2-mediated invasion in vitro and lung metastasis in vivo. We have demonstrated that SK-BR-3, a HER2-positive cell line, had a high level CCR4. In clinical samples, CCR4 expression was also associated with HER2 expression. These data indicated a potential link between CCR4 and HER2 expression in breast cancer. We treated SK-BR-3 cells with lapatinib, a HER2 antagonist, for different times and in different concentrations. No significant change of CCR4 expression in SK-BR-3 cells was observed (data not shown). The result showed that the inhibition of HER2 cannot downregulate CCR4 expression; many molecules in the cell transduction network could be involved in the interaction between CCR4 and HER2. Further study is needed to investigate the potential link between CCR4 and HER2 expression in breast cancer.

Molecular targeted therapy has become increasingly more important in the treatment of cancer. Anti-HER2 therapy has become a critical therapy in HER2 positive breast cancer [30]. KW-0761, a humanized anti-CCR4 antibody has been used as a new agent in the treatment of Adult T-cell leukemia/lymphoma [16]. In the present study, we have demonstrated that CCR4 can promote tumor growth and lung metastasis in breast cancer, and is associated with poor prognosis in patients with breast cancer. Olkhanud et al. [18] have showed eliminating CCR4-positive cells in 4T1 tumors can reduce lung metastasis in a murine model. Thus, targeting CCR4 could become a novel therapeutic strategy in breast cancer treatment in the future.

Acknowledgments

We thank the studied women for willingness to cooperate with our study. We thank prof. Ying Wang (Center for Human Disease Genomics, Peking University, Peking, China) for kindly providing the coding sequence of CCR4. This research is supported by the Shanghai United Developing Technology Project of Municipal Hospitals (SHDC12010116), Key Clinical Program of the Ministry of Health (2010-2012), and the National Natural Science Foundation of China (30971143, 30972936, 81001169).

Conflicts of interest

No potential conflicts of interest were disclosed.

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© Springer Science+Business Media, LLC. 2011