PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of neoplasiaGuide for AuthorsAbout this journalExplore this journalNeoplasia (New York, N.Y.)
 
Neoplasia. 2010 May; 12(5): 425–433.
PMCID: PMC2864480

Loss of Transforming Growth Factor-β Signaling in Mammary Fibroblasts Enhances CCL2 Secretion to Promote Mammary Tumor Progression through Macrophage-Dependent and -Independent Mechanisms1

Abstract

Whereas the accumulation of fibroblasts and macrophages in breast cancer is a well-documented phenomenon and correlates with metastatic disease, the functional contributions of these stromal cells on breast cancer progression still remain largely unclear. Previous studies have uncovered a potentially important role for CCL2 inflammatory chemokine signaling in regulating metastatic disease through a macrophage-dependent mechanism. In these studies, we demonstrate a significant regulatory mechanism for CCL2 expression in fibroblasts in mediating mammary tumor progression and characterize multiple functions for CCL2 in regulating stromal-epithelial interactions. Targeted ablation of the transforming growth factor-β (TGF-β) type 2 receptor in fibroblasts (Tgfbr2FspKO) results in a high level of secretion of CCL2, and cografts of Tgfbr2FspKO fibroblasts with 4T1 mammary carcinoma cells enhanced tumor progression associated with recruitment of tumor-associated macrophages (TAMs). Antibody neutralization of CCL2 in tumor-bearing mice inhibits primary tumor growth and liver metastases as evidenced by reduced cell proliferation, survival, and TAM recruitment. Both high and low stable expressions of small interfering RNA to CCL2 in Tgfbr2FspKO fibroblasts significantly reduce liver metastases without significantly affecting primary tumor growth, cell proliferation, or TAM recruitment. High but not low knockdown of CCL2 enhances tumor cell apoptosis. These data indicate that CCL2 enhances primary tumor growth, survival, and metastases in a dose-dependent manner, through TAM-dependent and -independent mechanisms, with important implications on the potential effects of targeting CCL2 chemokine signaling in the metastatic disease.

Introduction

The induction of host inflammatory responses toward breast cancer is characterized by the recruitment of stromal cells to the primary tumor, including macrophages and fibroblasts [1,2]. Accumulation of these stromal cells is associated with an increased expression of soluble factors, which alters the tumor microenvironment including extracellular matrix proteins, growth regulators, cytokines, and angiogenic factors, correlating with invasive breast cancer and poor patient prognosis [3,4]. Whereas the accumulation of fibroblasts and macrophages in breast cancer are a well-documented phenomenon, the signals that regulate the interplay between stromal and epithelial cancer cells remain complex and largely unclear.

A number of studies have indicated an important role for transforming growth factor-β (TGF-β) signaling in regulating fibroblast activation. TGF-β signaling activates mammary fibroblasts by inhibiting cell proliferation and inducing production of growth factors, angiogenic factors, extracellular matrix proteins, and proteases [5,6]. Whereas the role of autocrine TGF-β signaling in regulating the mesenchymal phenotype has been well documented [6,7], recent reports have indicated a significant role for TGF-β signaling in regulating interactions between stromal and epithelial cells. In recent studies, the role of TGF-β signaling in fibroblasts was analyzed by disrupting the expression of the TGF-β type 2 receptor (Tgfbr2) in a subset of fibroblasts by Cre-Lox (Tgfbr2FspKO). Co-transplanting mammary Tgfbr2FspKO fibroblasts with PyVmTor 4T1 mammary carcinoma cells significantly enhanced primary tumor growth and progression [8,9]. Whereas the protumorigenic phenotypes observed were associated with increased receptor tyrosine kinase signaling, including the hepatocyte growth factor/c-Met signaling pathway, abrogation of receptor tyrosine kinase signaling pathways did not completely inhibit tumor progression enhanced by the TGF-β signaling-deficient fibroblasts [8,9], indicating that TGF-β signaling regulates stromal-epithelial interactions through mechanisms independent of receptor tyrosine kinases.

The CCL2/CCR2 inflammatory chemokine signaling pathway has been shown to be important in regulating macrophage recruitment during inflammation and wound healing events [10,11]. Recent studies have demonstrated a potentially important role for CCL2 in regulating breast cancer progression in part through the recruitment of tumorassociated macrophages (TAMs). A high level of CCL2 expression in primary breast tumor has been shown to correlate with breast cancer invasiveness and decreased patient survival [12,13]. In mouse studies, a high level of expression of CCL2 in primary mammary tumors has also been associated with high levels of TAMs, cells that lack immunostimulatory function and secrete growth, survival, and angiogenic factors [14,15]. Tumor cells overexpressing recombinant CCL2 have been shown to recruit TAMs that enhance tumor growth and metastatic spread in mouse xenograft models of cancer [16,17], further implicating CCL2 in a protumorigenic role in breast cancer through a TAM-dependent mechanism.

This report shows that CCL2 and TGF-β signaling interacts to regulate stromal-epithelial interactions during mammary tumor progression. In these studies, Tgfbr2FspKO fibroblasts cografted with mammary carcinoma cells enhanced primary tumor growth and liver metastases statistically correlating with the accumulation of TAMs and increased expression of CCL2 in Tgfbr2FspKO fibroblasts. Antibody neutralization of CCL2 in tumor-bearing mice inhibited tumor growth and metastatic spread to the liver associated with reduced numbers of TAMs. Knockdown of CCL2 by stable small interfering RNA (siRNA) expression in Tgfbr2FspKO fibroblasts did not significantly affect primary tumor growth but did significantly reduce liver metastases and moderately reduce TAM recruitment. These data indicate that, whereas CCL2 in the breast cancer microenvironment functions to enhance primary tumor growth, survival, and metastases in part through TAM recruitment, CCL2 derived from mammary fibroblasts specifically functions to stimulate mammary carcinoma cells to promote cell survival and metastatic spread. In summary, these data are the first to demonstrate that TGF-β signaling in fibroblasts regulates metastatic disease through the CCL2 chemokine signaling pathway. Furthermore, these studies demonstrate a fibroblastspecific contribution of CCL2 to mammary tumor progression and are the first to demonstrate dose-dependent effects of CCL2 on primary tumor progression and TAM recruitment, with important implications on the potential effects of targeting CCL2 inflammatory chemokines signaling in metastatic disease.

Materials and Methods

Culture of Cell Lines

Floxed TGF-β type 2 receptor (Tgfbr2) control fibroblasts and Tgfbr2FspKO fibroblasts were immortalized and characterized in previous studies [8]. Fibroblasts, including stably expressing CCL2 siRNA cell lines, were cultured in Dulbecco's modified Eagle medium (DMEM)/F12/10% fetal bovine serum (FBS). 4T1 mammary carcinoma cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM/10% FBS. Phoenix cells were kindly provided by Jin Chen, MD, PhD, (Vanderbilt University, Nashville, TN) and were cultured in DMEM/10% FBS.

siRNA Silencing of CCL2 Expression in Tgfbr2FspKO Fibroblasts

The siRNA construct to target c-Met was obtained from Dr Martin Schwartz (University of Virginia, Charlottesville, VA). The H1 promoter and targeting sequences were digested from the previously mentioned constructs with HindIII and EcoRI and were cloned into the same sites of an siRNA expression vector (pSUPER) or an siRNA retroviral vector (pRETRO-SUPER). The pRETRO-SUPER vector was generously provided by Dr Reuven Agami (Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, the Netherlands) and described in Brummelkamp et al. [18]. The two targeting sequences used for siRNA mediated knockdown of CCL2 are 5′-CAGAACCTACAACTTTATT-3′ for 1CCL2- and 5′-TAAATCTGAAGCTAATGCA-3′ for 3CCL2-. The targeting sequence to silence enhanced GFP (eGFP) as a negative control is 5′-GCTGACCCTGAAGTTCATC-3′. 1CCL2-, 3CCL2-, and eGFP targeting oligonucleotides were designed as previously described [18]. The oligonucleotides were phosphorylated by kinase treatment; complementary oligos were then annealed and subcloned into the BglII and HindIII sites of pRETRO-SUPER. Plasmids were transfected into Phoenix cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Forty-eight hours after transfection, Tgfbr2FspKO fibroblasts were transduced with virus-conditioned medium and selected with 1.5 µg/ml puromycin.

Mouse Strains and Maintenance

Female nude (nu/nu) mice (6–8 weeks of age) were obtained from Harlan Laboratories (Denver, CO). The experimental research on rodents reported here has been performed with the approval of the appropriate ethics committees, including the Association for Assessment and Accreditation of Laboratory Animal Care and University of Kansas Institutional Animal Care and Use Committee.

Subrenal Capsule Grafting

Grafting of collagen-embedded cells was performed according to the methods of Hayward et al. [19]. Briefly, 1 x 105 4T1 cells were resuspended together with 2.5 x 105 Tgfbr2FspKO or Tgfbr2Flox/Flox fibroblasts in 50 µl of collagen per graft. The collagen-embedded cells were cultured in DMEM/F12 10% FBS for 24 hours and then implanted under the renal capsule layer of the kidneys in female nude mice, 6 to 8 weeks of age. Neutralizing antibodies that recognize murine-specific CCL2 (R&D Systems, Minneapolis, MN) or control immunoglobulin G (IgG; Sigma, St Louis, MO) were solubilized in 0.9% saline and injected into the intraperitoneum into mice 7 days after grafting at 5 mg/kg, every 2 days for 14 days. Tumor tissues were collected 21 days after implantation and weighed. Liver metastases were counted by examination of gross tissues. Metastatic lesions on the liver were confirmed by hematoxylin and eosin stain as previously shown [9].

Histology and Immunohistochemistry

Tumor tissues were fixed in 10%neutral formalin buffer, subjected to dehydration in 50%, 70%, 80%, 95%, and 100% ethanols and xylene and then paraffin-embedded. Five-micrometer sections were prepared for immunohistochemistry. The sections were stained with hematoxylin and eosin. Immunofluorescence staining of F4/80 (rat monoclonal; Abcam, Cambridge, MA) was performed by 10 nM sodium citrate antigen retrieval for 10 minutes at 105°C and by incubating primary antibodies at a 1:50 dilution overnight at 4°C. Sections were incubated with rat biotinylated secondary antibodies at a 1:500 dilution, conjugated to streptavidin-Alexa 568 (1:500; Molecular Probes, Invitrogen, Carlsbad, CA) and counterstained with 4′6′-diamidino-2-phenylindole. Sections were mounted with Prolong Antifade (Molecular Probes). Immunoperoxidase staining for cleaved caspase-3 (rabbit polyclonal; Cell Signaling Technologies, Boston, MA) and Ki67 (rabbit polyclonal; Dako, Carpinteria, CA) was performed by citrate antigen retrieval and by incubating primary antibodies at a 1:100 dilution overnight at 4°C. Sections were incubated with appropriate secondary biotinylated antibodies at a 1:500 dilution and conjugated to streptavidin peroxidase (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA) according to a commercial protocol. Sections were visualized by peroxidase staining (Vectastain EliteKit; Vector Laboratories) and counterstained with hematoxylin. Proliferative and apoptotic indices were calculated by determining the relative area of positively stained cells to the total number of cells in at least five high-powered fields using Scion Image software (Frederick, MA).

Flow Cytometry Analysis

Primary tumor tissues were digested into single-cell suspensions according to previously described studies [20]. Briefly, tumor tissues were digested in PBS buffer containing 0.4 mg/ml collagenase, 2 mg/ml hyaluronidase, and 2 mg/ml trypsin and separated into a single-cell suspension using a manual tissue homogenizer. Debris were removed by passing through a mesh filter, and red blood cells were lysed using a buffer containing 10 mM KHCO3, 150 mM NH4Cl, and 0.1 mM EDTA. Samples were incubated with anti-F4/80-PE (AbdSerotec, Oxford, UK), anti-Cd11b-FITC, and anti-Gr1-APC (BD Pharmingen, San Jose, CA), 1:100 on ice, washed with PBS, and then analyzed by flow cytometry using a BD FACS analyzer (BD Biosciences, San Jose, CA).

Determination of CCL2 Levels in Fibroblasts

To determine the levels of CCL2 secreted from fibroblasts in culture by ELISA, immortalized and primary cultures of fibroblasts from Tgfbr2Flox/Flox and Tgfbr2FspKO mammary glands were grown in 10-cm plates in DMEM/F12 medium containing 10% FBS and antibiotics. At 80% confluence, the cells were starved in DMEM F12 medium containing 0.1% FBS for 24 hours and were then incubated in 4 ml of complete medium for another 24 hours. Levels of secreted CCL2 protein were determined by ELISA (R&D Systems) using 100 µl of conditioned medium. Statistical significance was determined by two-tailed Student's t test.

Determination of Anti-CCL2 and IgG Levels in Tumor-Bearing Mice

To determine the levels of anti-CCL2 and control IgG in tumor-bearing mice by ELISA, blood sera were collected from the tail vein of grafted mice 4 and 24 hours after the last treatment with anti-CCL2 or IgG. The samples were then diluted in buffer containing 50 mM NaHCO3 and 50 mM Na2CO3, pH9.6, and coated onto 96-well plates overnight at room temperature. The samples were incubated with antirat biotinylated antibodies (1:500) for 1 hour and then with streptavidin peroxidase (Vectastain Elite Kit; Vector Laboratories) for an additional 30 minutes. The samples were visualized by incubating with tetramethylbenzidine substrate (R&D Systems) according to the manufacturer's instructions. The reaction was stopped with 1MHCl, and the absorbance was read at OD 450 nm.

Statistical Analyses

Data were analyzed for statistical significance by two-tailed Student's t test, ANOVA with Bonferroni's multiple comparison tests of all groups, or Spearman rank correlation test as indicated, using GraphPad Prism software (GraphPad Software, La Jolla, CA). Statistical significance was determined by P < 0.05.

Results

Tgfbr2FspKO Fibroblasts Enhance Recruitment of TAMs to the Primary Tumor Associated with Increased Expression of CCL2

In previous studies, Tgfbr2-deficient fibroblasts cografted with mammary carcinoma cells [8,9] resulted in enhanced primary tumor growth and invasiveness and metastatic spread. Studies have shown that accumulation of immune cells, in particular, TAMs, correlates with the invasive phenotype of breast cancers; therefore, it was important to determine whether Tgfbr2-deficient fibroblasts significantly affected recruitment of macrophages to the primary tumor. Tgfbr2FspKO fibroblasts were cografted with 4T1 mammary carcinoma cells in the subrenal capsule of nude mice for 21 days and subjected to flow cytometry analysis for a number of cells coexpressing F4/80 and Cd11b, markers associated with TAMs [14]. Tgfbr2FspKO fibroblasts cografted with 4T1 mammary carcinoma cells resulted in significantly higher levels of F4/80, Cd11b double-positive macrophages compared with mammary carcinoma cells cografted with control Tgfbr2Flox/Flox fibroblasts (Figure 1, A and B). Furthermore, correlation analyses revealed a significant association between the increased numbers of F4/80, Cd11b-positive cells recruited to the primary tumor, with the increased primary tumor growth and liver metastases of mammary carcinoma cells cografted with Tgfbr2FspKO fibroblasts (Figure 1, C and D).

Figure 1
Tgfbr2FspKO fibroblasts enhance recruitment of TAMs. (A) Primary tumor grafts were stained by immunofluorescence for F4/80 expression to detect TAMs. Sections were counterstained with DAPI. Arrows point to F4/80-positive cells. Scale bar, 40 ε ...

Previous studies have shown that macrophage recruitment in part is regulated by the expression of chemokines in the tissue microenvironment [16,21]. In particular, CCL2 was previously shown a significant regulator of macrophage recruitment during inflammation and tumorigenesis [1,22]. By Affymetrix complementary DNA microarray analysis, CCL2 was observed to be the most significantly upregulated of all chemokines in Tgfbr2FspKO fibroblasts, in comparison with control fibroblasts (data not shown). This upregulated expression was confirmed by ELISA of supernatants isolated from cultured primary and immortalized Tgfbr2FspKO and control Tgfbr2Flox/Flox fibroblasts (Figure 2A). Furthermore, primary tumors cografted with Tgfbr2FspKO fibroblasts also showed high levels of CCL2 expression localized to the stromal fibroblast compartment, which was absent in the stromal tissues of primary tumors cografted with control fibroblasts (Figure 2B). These data indicated an association between enhanced CCL2 expression in Tgfbr2FspKO fibroblasts and recruitment of TAMs, leading to the hypothesis that CCL2 derived from TGF-β signaling-deficient fibroblasts enhances mammary tumor progression through TAM recruitment.

Figure 2
Increased expression of CCL2 in Tgfbr2FspKO stromal cells. (A) Secretion of CCL2 from fibroblast conditioned medium from primary and immortalized Tgfbr2Flox/Flox and Tgfbr2FspKO fibroblasts was quantified by ELISA. Statistical significance was determined ...

Antibody Neutralization of CCL2 Inhibits Mammary Tumor Progression and TAM Recruitment

To determine the functional contribution of CCL2 derived from Tgfbr2FspKO fibroblasts in mammary tumorigenesis, two approaches were used to inhibit CCL2 activity. First, tumor-bearing mice were treated with neutralizing antibodies to CCL2, which has been previously shown to inhibit CCL2-induced inflammation in mice [23,24]. Tgfbr2FspKO fibroblasts were cografted with 4T1 mammary carcinoma cells in the subrenal capsule of nude mice. Seven days after treatment, grafted mice were injected in the intraperitoneum with 5 mg/kg of anti-CCL2, control IgG, or 0.9% saline vehicle control for an additional 14 days. To determine the stability of anti-CCL2 injected in mice, samples of blood serum were collected from mice by tail vein and were analyzed for levels of anti-CCL2 4 and 24 hours before the last injection. ELISA of blood serum indicated significant levels of anti-CCL2 24 hours after injection, although the levels were lower compared with the levels of control rat IgG (Figure 3A). Compared with IgG controls, anti-CCL2 treatment resulted in a significant reduction in primary mammary tumor mass (Figure 3B) associated with decreased TAM recruitment to the primary tumor (Figure 3C). In addition, anti-CCL2 treatment also significantly decreased the number but not the incidence of liver metastases compared with rat IgG- or saline-treated mice (Figure 3D). These data indicate that systemic inhibition of CCL2 significantly inhibits tumor growth and metastatic spread associated with TAM recruitment.

Figure 3
Effect of CCL2 neutralization on primary tumor and recruitment of TAMs. (A) Peripheral blood samples were harvested from tumor-bearing mice 4 and 24 hours after injection and analyzed for the presence of anti-CCL2 or control IgG by ELISA. (B) Primary ...

Immunohistochemistry analyses were performed on primary tumor sections to determine the effects of anti-CCL2 treatment on tumor cell proliferation and survival. Anti-CCL2-treated tumors exhibited significant decreases in cellular proliferation as indicated by K67 staining (Figure 4A) compared with IgG- and saline-treated mice. Whereas anti-CCL2 treatment resulted in a significantly increased cleaved caspase-3 staining compared with saline-treated groups, there were no significant differences in cleaved caspase-3 expression between IgG- and anti-CCL2- treated groups because IgG-treated groups also exhibited increased apoptosis compared with saline (Figure 4B). In summary, these data indicate that the reduction in cellular proliferation and cell survival in anti-CCL2-treated mice was associated with an overall reduction in primary tumor growth and liver metastases.

Figure 4
Effect of anti-CCL2 treatment on tumor cell proliferation and apoptosis. Primary tumor graft recombinants were paraffin-embedded, sectioned, and immunostained for the expression of (A) Ki67 as a marker for cell proliferation and (B) cleaved caspase-3 ...

siRNA Silencing of CCL2 in Tgfbr2FspKO Fibroblasts Inhibits Liver Metastases but Does Not Significantly Reduce TAM Recruitment

Studies have shown that other cell types in the tumor microenvironment including macrophages, endothelial cells, and mammary epithelial cells expressed CCL2 [25,26]. To determine the functional contribution of CCL2 derived from Tgfbr2 deficient fibroblasts, an siRNA approach to target CCL2 expression was used. siRNA targeting two different regions of the Ccl2 transcript were stably expressed in Tgfbr2FspKO fibroblasts, resulting in the generation of two cell lines, one exhibiting 35% knockdown (3CCL2.) and the other exhibiting 82% knockdown (1CCL2-) of CCL2 expression compared with control Tgfbr2FspKO fibroblasts expressing siRNA to GFP (GFP-) (Figure 5A). Cografting 4T1 mammary carcinoma cells with fibroblast cell lines expressing varying levels of CCL2 allowed the determination of dose-dependent effects of CCL2 derived from mammary fibroblasts on mammary tumor progression. Compared with cografting 4T1 cells with GFP. control fibroblasts, cografting of 3CCL2- fibroblasts did not result in significant changes to tumor mass (Figure 5B), TAM recruitment (Figure 5C), or liver metastases (Figure 5D). These data indicate that a 35% knockdown in CCL2 secretion in Tgfbr2FspKO fibroblasts was not sufficient to inhibit mammary tumor growth, TAM recruitment, or metastatic spread. Cografting with 1CCL2- fibroblasts did not result in significant changes to primary tumor growth (Figure 5B). We observed a small decrease in TAM recruitment, which was not statistically significant (Figure 5C). In addition, cografting with 1CCL2- fibroblasts resulted in a significant decrease in the number but not in the incidence of liver metastases (Figure 5D). These data indicate that CCL2 derived from Tgfbr2FspKO fibroblasts does not significantly regulate primary tumor growth or TAM recruitment but does, in part, regulate liver metastases of 4T1 mammary carcinoma cells, possibly in a concentration-dependent manner.

Figure 5
Effect of siRNA knockdown in Tgfbr2FspKO fibroblasts on 4T1 tumor growth and recruitment of TAMs. (A) Conditioned medium from parental fibroblasts (Par), fibroblasts stably expressing control siRNA (GFP-), or siRNA targeting CCL2 (3CCL2- and 1CCL2-) were ...

Immunohistochemistry analyses were performed to determine the effects of inhibiting CCL2 by knockdown in fibroblasts on primary tumor cell proliferation and survival. Transplantation of 4T1 mammary carcinoma cells with 3CCL2- fibroblasts did not result in significant changes in Ki67 staining or cleaved caspase-3 staining (Figure 6, A and B). Cografting of 4T1 carcinoma cells with 1CCL2- fibroblasts also did not result in changes to Ki67 staining but did result in significantly increased cleaved caspase-3 staining. These data indicate that CCL2 derived from Tgfbr2FspKO fibroblasts does not regulate tumor cell proliferation but does regulate tumor cell survival, possibly in a concentration-dependent manner.

Figure 6
Effect of CCL2 knockdown in fibroblasts on tumor cell proliferation and survival. Immunostaining of primary sections of control, 3CCL2-, and 1CCL2- cocultures for the expression of Ki67 as a marker for cell proliferation (A) and of cleaved caspase-3 as ...

Discussion

Previous studies have shown that TGF-β signaling in fibroblasts suppresses mammary carcinoma cell growth, invasion, and metastatic spread by regulating expression of soluble factors that mediate tyrosine receptor kinase signaling in mammary carcinoma cells [8]. In these present studies, the data collectively support that TGF-β signaling in mammary fibroblasts inhibits CCL2 chemokine expression to negatively regulate TAM recruitment and tumor metastases. These studies reveal important insight into the relationship between TGF-β and CCL2 signaling pathways during breast cancer progression and reveal important roles for CCL2 in regulating fibroblast interactions with mammary carcinoma cells and other stromal cells in the mammary tumor microenvironment.

Whereas previous studies have demonstrated that CCL2 regulates breast cancer progression through TAM recruitment, the effects of siRNA knockdown in CCL2 in mammary fibroblasts presented here indicate that CCL2 regulates breast cancer progression in part by mediating fibroblast-carcinoma cell interactions. We observed that a 35% knockdown in CCL2 expression in Tgfbr2FspKO fibroblasts was not sufficient to inhibit macrophage recruitment, although a slight reduction in the number of liver metastases was noted. An 82% knockdown in CCL2 expression resulted in a small but not statistically significant reduction in macrophage recruitment but did significantly reduce liver metastases and cell survival. The different effects of CCL2 knockdown in fibroblasts on macrophage recruitment and 4T1 metastases indicate that CCL2 secreted from Tgfbr2FspKO fibroblasts acts directly on mammary carcinoma cells to regulate cell survival and metastatic spread (Figure 7A). Interestingly, a recent study demonstrated a different mechanism through which CCL2 may regulate the metastatic spread. This particular study demonstrated that overexpression of CCL2 in prostate cancer cells resulted in increased bone metastases in mice, associated with increased osteoclast formation, indicating that CCL2 may act directly or indirectly on stromal cells of distal sites to regulate metastatic spread [16]. In summary, these studies indicate that CCL2 may regulate late-stage carcinoma progression at the primary tumor site and in distal sites of metastasis, through complex, multiple mechanisms.

Figure 7
Model for the role of CCL2 signaling in the breast tumor microenvironment. (A) Mammary fibroblasts increase CCL2 expression when TGF-β signaling is downregulated. The increased expression of CCL2 derived from mammary fibroblasts acts directly ...

Currently, the functional contribution of CCL2/CCR2 signaling in cancer cells remains largely unclear. The polymorphism CCR264-l has been associated with the development sporadic breast cancer [27], and CCR2 overexpression significantly correlates with the development of glioblastoma [28]. Studies of CCL2 signaling in the human PC-3 prostate cancer cell line demonstrate that CCL2 promotes cell survival through an AKT-independent mechanism, by negatively regulating AMP-activated protein kinase resulting in mammalian target of rapamycin complex activation and sustained expression of survivin [29]. In addition, current studies in our laboratory indicate that CCL2/CCR2 signaling regulates mammary carcinoma cell survival and invasion through a mitogen-activated protein kinase-dependent mechanism (unpublished observations). These studies indicate that CCL2 promotes cell survival in different cancer cell types, potentially through multiple mechanisms. Further studies are underway in our laboratory to further understand the molecular mechanisms through which CCL2 mediates stromal-breast cancer cell interactions.

An important association between TAM recruitment and CCL2 in mammary tumor progression was also observed in these current studies. Given that anti-CCL2 antibody inhibited mammary tumor progression more significantly than siRNA knockdown of CCL2 in mammary fibroblasts, it is possible that CCL2 derived from TAMs also promotes primary tumor growth and TAM recruitment (Figure 7B). Anti-CCL2 treatment but not siRNA knockdown of CCL2 in fibroblasts inhibited primary tumor growth, indicating that systemic inhibition of CCL2 expressed in other cell types may also contribute to mammary tumor growth. Because recombinant CCL2 does not significantly affect cellular proliferation in vitro (data not shown), CCL2 may enhance primary tumor growth, indirectly through enhancement of TAMs that have been shown to express a number of growth factors [1,30]. This possibility is also supported in previous studies, in which anti-CCL2 treatment of tumor-bearing mice inhibited growth and metastases of MCF7 and MDA-231 cells orthotopically grafted in severe combined immunodeficient mice along with a decrease in macrophage recruitment [17,31]. These studies further suggested that CCL2 expression was derived from TAMs that were recruited to the primary tumor through a CCL2-dependent positive feedback mechanism [31]. CCL2-dependent recruitment of TAMs to the primary tumor has also been demonstrated in a transplantation model of prostate cancer [16]. Anti-CCL2 treatment of prostate tumor-bearing mice inhibited tumor growth associated with decreased macrophage recruitment, indicating that CCL2 may regulate tumor progression through a macrophage-dependent mechanism in multiple tumor types. Because Tgfbr2FspKO fibroblasts cografted with mammary carcinoma cells resulted in enhanced recruitment of TAMs, it is possible that the TAM recruitment was initiated by Tgfbr2FspKO fibroblasts, subsequently resulting in a CCL2-dependent positive feedback mechanism to recruit additional TAMs over time. The data presented here support an important association between CCL2 and TAM recruitment in metastatic disease. Collectively, these studies demonstrate multifunctional roles for CCL2 signaling in breast cancer and indicate that targeting CCL2 in breast cancer would inhibit both epithelial and mesenchymal cell types.

Studies presented here indicate that differing levels of CCL2 in the tumor microenvironment significantly affect the activity of stromal cells and mammary carcinoma cells. Cografting 4T1 cells with 1CCL2-fibroblasts but not 3CCL2- fibroblasts significantly inhibited liver metastases. In addition, cografting experiments with 1CCL2- fibroblasts but not with 3CCL2- fibroblasts resulted in a small decrease in macrophage recruitment, indicating that local concentrations of CCL2 may stimulate stromal cells at levels different from mammary carcinoma cells. This notion is supported in previous studies that show that high concentrations of CCL2 in mammary tumors desensitize and downregulate chemokine receptor signaling of T cells while supporting tumor growth [32]. Although CCL2 has been found to bind and activate CCR2 in mouse and human macrophages with kd values of 22.5 and 25.7 nM, respectively [33], this study, as well as previous studies [32], indicates that CCL2/CCR2 binding kinetics differ among mammary epithelial cells or other mesenchymal cell types. Given that chemokine receptor desensitization and down-regulation is a common mechanism in regulating G-coupled receptor signaling in multiple cell types [32,34,35], it would be important in the future to clarify and understand the signaling kinetics of CCL2 chemokine signaling among stromal cells and carcinoma cells to predict its biological effects on tumor progression.

This study of siRNA knockdown in CCL2 in fibroblasts demonstrates a fibroblast-specific contribution of CCL2 signaling in regulating mammary tumor progression. In addition, data presented here support the potential effectiveness for targeting the CCL2/CCR2 network in metastatic disease. However, given that CCL2 acts on multiple cell types in the tumor microenvironment and that chemokine signaling is dependent on ligand concentrations, it is important to clarify the molecular and cellular mechanisms of inflammatory chemokine signaling in breast cancer to design an effective therapeutic regimen for the treatment of metastatic disease.

Acknowledgments

The authors thank Wei Bin Fang (University of Kansas Medical Center), Karen Stunk (University of North Carolina, Chapel Hill), and Rebecca Cook (Vanderbilt University, Nashville, TN) for the critical reading of the manuscript. The authors also thank the Flow Cytometry Core Facility for assistance with the flow cytometry studies and the Biostatistical Core Facility at the Kansas University Medical Center for assistance in statistical analysis of data.

Abbreviations

siRNA
small interfering RNA
TAM
tumor-associated macrophage
Tgfbr2
type 2TGF-β receptor gene
Tgfbr2Flox/Flox
Tgfbr2 with loxP sites flanking exon 2
Tgfbr2FspKO
conditional knockout of Tgfbr2 by Fsp-1 Cre

Footnotes

1These studies were supported by funds from the National Cancer Institute/National Institutes of Health 1K99CA127357-01A2 (N.C.) and University of Kansas Endowment.

The authors declare that no competing interests exist.

References

1. Allavena P, Sica A, Solinas G, Porta C, Mantovani A. The inflammatory micro-environment in tumor progression: the role of tumor-associated macrophages. Crit Rev Oncol Hematol. 2008;66:1–9. [PubMed]
2. Bierie B, Moses HL. Under pressure: stromal fibroblasts change their ways. Cell. 2005;123:985–987. [PubMed]
3. Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, Chen H, Omeroglu G, Meterissian S, Omeroglu A, et al. Stromal gene expression predicts clinical outcome in breast cancer. Nat Med. 2008;14:518–527. [PubMed]
4. Bieche I, Lerebours F, Tozlu S, Espie M, Marty M, Lidereau R. Molecular profiling of inflammatory breast cancer: identification of a poor-prognosis gene expression signature. Clin Cancer Res. 2004;10:6789–6795. [PubMed]
5. Abraham S, Sawaya BE, Safak M, Batuman O, Khalili K, Amini S. Regulation of MCP-1 gene transcription by Smads and HIV-1 Tat in human glial cells. Virology. 2003;309:196–202. [PubMed]
6. Akhurst RJ, Derynck R. TGF-B signaling in cancer—a double edged sword. Trends Cell Biol. 2001;11:S44–S50. [PubMed]
7. Wells RG, Discher DE. Matrix elasticity, cytoskeletal tension, and TGF-β: the insoluble and soluble meet. Sci Signal. 2008;1:pe13. [PMC free article] [PubMed]
8. Cheng N, Bhowmick NA, Chytil A, Gorksa AE, Brown KA, Muraoka R, Arteaga CL, Neilson EG, Hayward SW, Moses HL. Loss of TGF-β type II receptor in fibroblasts promotes mammary carcinoma growth and invasion through upregulation of TGF-β-, MSP- and HGF-mediated signaling networks. Oncogene. 2005;24:5053–5068. [PMC free article] [PubMed]
9. Cheng N, Chytil A, Shyr Y, Joly A, Moses HL. Enhanced hepatocyte growth factor signaling by type II transforming growth factor-{beta} receptor knockout fibroblasts promotes mammary tumorigenesis. Cancer Res. 2007;67:4869–4877. [PubMed]
10. Boring L, Gosling J, Chensue SW, Kunkel SL, Farese RV, Jr, Broxmeyer HE, Charo IF. Impaired monocyte migration and reduced type 1 (TH1) cytokine responses in C-C chemokine receptor 2 knockout mice. J Clin Invest. 1997;100:2552–2561. [PMC free article] [PubMed]
11. Kurihara T, Warr G, Loy J, Bravo R. Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor. J ExpMed. 1997;186:1757–1762. [PMC free article] [PubMed]
12. Valkovic T, Lucin K, Krstulja M, Dobi-Babic R, Jonjic N. Expression of monocyte chemotactic protein-1 in human invasive ductal breast cancer. Pathol Res Pract. 1998;194:335–340. [PubMed]
13. Soria G, Yaal-Hahoshen N, Azenshtein E, Shina S, Leider-Trejo L, Ryvo L, Cohen-Hillel E, Shtabsky A, Ehrlich M, Meshel T, et al. Concomitant expression of the chemokines RANTES and MCP-1 in human breast cancer: a basis for tumor-promoting interactions. Cytokine. 2008;44:191–200. [PubMed]
14. Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer Res. 2006;66:605–612. [PubMed]
15. Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell. 2006;124:263–266. [PubMed]
16. Mizutani K, Sud S, McGregor NA, Martinovski G, Rice BT, Craig MJ, Varsos ZS, Roca H, Pienta KJ. The chemokine CCL2 increases prostate tumor growth and bone metastasis through macrophage and osteoclast recruitment. Neoplasia. 2009;11:1235–1242. [PMC free article] [PubMed]
17. Lu X, Kang Y. Chemokine (C-C motif) ligand 2 engages CCR2+ stromal cells of monocytic origin to promote breast cancermetastasis to lung and bone. J Biol Chem. 2009;284:29087–29096. [PMC free article] [PubMed]
18. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science. 2002;296:550–553. [PubMed]
19. Hayward S, Haughney PC, Rosen MA, Greulich KM, Weier HU, Dahiya R, Cunha GR. Interactions between adult human prostatic epithelium and rat urogenital sinus mesenchyme in a tissue recombination model. Differentiation. 1998;63:131–140. [PubMed]
20. Yang L, DeBusk LM, Fukuda K, Fingleton B, Green-Jarvis B, Shyr Y, Matrisian LM, Carbone DP, Lin PC. Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell. 2004;6:409–421. [PubMed]
21. Lu B, Rutledge BJ, Gu L, Fiorillo J, Lukacs NW, Kunkel SL, North R, Gerard C, Rollins BJ. Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J Exp Med. 1998;187:601–608. [PMC free article] [PubMed]
22. De Paepe B, Creus KK, De Bleecker JL. Chemokines in idiopathic inflammatory myopathies. Front Biosci. 2008;13:2548–2577. [PubMed]
23. Brydon EW, Smith H, Sweet C. Influenza A virus-induced apoptosis in bronchiolar epithelial (NCI-H292) cells limits pro-inflammatory cytokine release. J Gen Virol. 2003;84:2389–2400. [PubMed]
24. Christophi GP, Hudson CA, Panos M, Gruber RC, Massa PT. Modulation of macrophage infiltration and inflammatory activity by the phosphatase SHP-1 in virus-induced demyelinating disease. J Virol. 2009; 83:522–539. [PMC free article] [PubMed]
25. Salcedo R, Ponce ML, Young HA, Wasserman K, Ward JM, Kleinman HK, Oppenheim JJ, Murphy WJ. Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression. Blood. 2000;96:34–40. [PubMed]
26. Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, Koike M, Inadera H, Matsushima K. Significance of macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin Cancer Res. 2000;6:3282–3289. [PubMed]
27. Zafiropoulos A, Crikas N, Passam AM, Spandidos DA. Significant involvement of CCR2-64I and CXCL12-3a in the development of sporadic breast cancer. J Med Genet. 2004;41:e59. [PMC free article] [PubMed]
28. Liang Y, Bollen AW, Gupta N. CC chemokine receptor-2A is frequently overexpressed in glioblastoma. J Neurooncol. 2008;86:153–163. [PubMed]
29. Roca H, Varsos ZS, Pienta KJ. CCL2 is a negative regulator of AMP-activated protein kinase to sustain mTOR complex-1 activation, survivin expression, and cell survival in human prostate cancer PC3 cells. Neoplasia. 2009;11:1309–1317. [PMC free article] [PubMed]
30. Porta C, Subhra Kumar B, Larghi P, Rubino L, Mancino A, Sica A. Tumor promotion by tumor-associated macrophages. Adv Exp Med Biol. 2007;604:67–86. [PubMed]
31. Fujimoto H, Sangai T, Ishii G, Ikehara A, Nagashima T, Miyazaki M, Ochiai A. Stromal MCP-1 in mammary tumors induces tumor-associated macrophage infiltration and contributes to tumor progression. Int J Cancer. 2009;125:1276–1284. [PubMed]
32. Kurt RA, Baher A, Wisner KP, Tackitt S, Urba WJ. Chemokine receptor desensitization in tumor-bearing mice. Cell Immunol. 2001;207:81–88. [PubMed]
33. Wang JM, Hishinuma A, Oppenheim JJ, Matsushima K. Studies of binding and internalization of human recombinant monocyte chemotactic and activating factor (MCAF) by monocytic cells. Cytokine. 1993;5:264–275. [PubMed]
34. Devalaraja MN, Richmond A. Multiple chemotactic factors: fine control or redundancy? Trends Pharmacol Sci. 1999;20:151–156. [PubMed]
35. Ozawa S, Kato Y, Komori R, Maehata Y, Kubota E, Hata R. BRAK/CXCL14 expression suppresses tumor growth in vivo in human oral carcinoma cells. Biochem Biophys Res Commun. 2006;348:406–412. [PubMed]

Articles from Neoplasia (New York, N.Y.) are provided here courtesy of Neoplasia Press