Immunology and Cell Biology (2014) 92, 815–824 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb

ORIGINAL ARTICLE

The atypical chemokine receptor CCX-CKR regulates metastasis of mammary carcinoma via an effect on EMT Yuka Harata-Lee1, Michelle E Turvey1, Julie A Brazzatti1,5, Carly E Gregor1, Michael P Brown2, Mark J Smyth3,4, Iain Comerford1 and Shaun R McColl1 Over the last decade, the significance of the homeostatic CC chemokine receptor-7 and its ligands CC chemokine ligand-19 (CCL19) and CCL21, in various types of cancer, particularly mammary carcinoma, has been highlighted. The chemokine receptor CCX-CKR is a high-affinity receptor for these chemokine ligands but rather than inducing classical downstream signalling events promoting migration, it instead sequesters and targets its ligands for degradation, and appears to function as a regulator of the bioavailability of these chemokines in vivo. Therefore, in this study, we tested the hypothesis that local regulation of chemokine levels by CCX-CKR expressed on tumours alters tumour growth and metastasis in vivo. Expression of CCX-CKR on 4T1.2 mouse mammary carcinoma cells inhibited orthotopic tumour growth. However, this effect could not be correlated with chemokine scavenging in vivo and was not mediated by host adaptive immunity. Conversely, expression of CCX-CKR on 4T1.2 cells resulted in enhanced spontaneous metastasis and haematogenous metastasis in vivo. In vitro characterisation of the tumourigenicity of CCX-CKR-expressing 4T1.2 cells suggested accelerated epithelial–mesenchymal transition (EMT) revealed by their more invasive and motile character, lower adherence to the extracellular matrix and to each other, and greater resistance to anoikis. Further analysis of CCX-CKR-expressing 4T1.2 cells also revealed that transforming growth factor (TGF)-b1 expression was increased both at mRNA and protein levels leading to enhanced autocrine phosphorylation of Smad 2/3 in these cells. Together, our data show a novel function for the chemokine receptor CCX-CKR as a regulator of TGF-b1 expression and the EMT in breast cancer cells. Immunology and Cell Biology (2014) 92, 815–824; doi:10.1038/icb.2014.58; published online 15 July 2014

INTRODUCTION Chemokines comprise a large family of functionally and structurally related proteins with a plethora of biological functions. Although best characterised for their critical role in directing leukocyte migration, chemokines have been implicated in other biological processes such as angiogenesis, proliferation, regulation of apoptosis and cell adhesion.1–3 Consistent with these activities, growing evidence indicates that some chemokines promote the growth and metastasis of various cancers.4,5 More specifically, the homeostatic chemokine receptor CC chemokine receptor-7 (CCR7) has been shown to be upregulated in human mammary tumours compared with normal tissues.6 The same study also demonstrated that the chemokines CC chemokine ligand (CCL)-19 and CCL21, the ligands of CCR7, are highly expressed in human organs to which breast cancer is known to metastasise. In addition, studies conducted in our laboratory

1Chemokine

indicated that CCL21 promotes metastasis of human breast cancer cells by rendering tumour cells more resistant to anoikis.7 On the other hand, contrary to these observations, several groups have also demonstrated a role for CCL19, CCL21 or CCR7 in driving the host immune response against various types of tumours.8–12 These studies suggest that the effect of CCL19, CCL21 and CCR7 on cancer progression may be context dependent. Recently, several chemokine receptors have been identified that differ from all other known chemokine receptors in several important aspects, leading to their classification as ‘atypical’ chemokine receptors. This family includes DARC, D6 and CCX-CKR. These receptors, although structurally resembling other chemokine receptors and binding to ligands with high affinity, do not couple to signalling cascades that promote cell migration following chemokine ligation. This has led to the proposition that they may act as chemokine decoy

Biology Laboratory, Centre for Molecular Pathology, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia; Therapeutics Laboratory, Cancer Clinical Trials Unit, Royal Adelaide Hospital, Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia; 3Immunology in Cancer and Infection Laboratory, Department of Immunology, Queensland Institute of Medical Research, Herston, Queensland, Australia and 4School of Medicine, University of Queensland, Herston, Queensland, Australia 5Current Address: Stem Cell and Leukaemia Proteomics Lab, Institute of Cancer Sciences, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK. Correspondence: Professor SR McColl, Centre for Molecular Pathology, School of Molecular and Biomedical Science, University of Adelaide, Molecular Life Sciences Building North Terrace, Adelaide, 5005 South Australia, Australia. E-mail: [email protected] Received 27 October 2013; revised 16 May 2014; accepted 6 June 2014; published online 15 July 2014 2Experimental

Novel function of CCX-CKR in mammary carcinoma Y Harata-Lee et al 816

or transport receptors.13 One of these receptors CCX-CKR binds to the CCR7 ligands CCL19 and CCL21, and has been shown in vitro to internalise and degrade these ligands14–16 and also to control the abundance of these chemokines in vivo with consequent effects on the development of immune system and regulation of host immune responses.17,18 Despite significant evidence suggesting an important role for the CCX-CKR ligands, CCL19 and CCL21 and their signalling receptor CCR7 in progression and metastasis of mammary carcinoma, the role of CCX-CKR in tumour biology has not been extensively investigated in the past. Feng and colleagues examined the effect of CCX-CKR expression on a human breast cancer cell line using an athymic mouse model. Although they showed inhibition of primary tumour growth and metastasis and a negative correlation between CCX-CKR expression in primary tumours and lymph node metastasis,19 the mechanisms by which CCX-CKR expression led to these effects were not determined. Therefore, in this study, we have investigated the effect of CCX-CKR expression on mammary carcinoma using a syngeneic mouse model to elucidate how CCX-CKR affects mammary tumour progression and metastasis. RESULTS Expression of CCX-CKR enhances metastasis of 4T1.2 mammary tumours in vivo while inhibiting primary tumour growth In order to investigate the role of CCX-CKR on progression and metastasis of breast cancer, a plasmid encoding mouse CCX-CKR or the empty plasmid were stably transfected into murine 4T1.2 mammary carcinoma cells (4TCCX or 4TpEF). Expression of CCXCKR by 4TCCX was confirmed by quantitative PCR (qPCR; Supplementary Figure 1a), and its effect on ligand scavenging by 4T1.2 cells was tested by incubating these cells in medium containing the CCX-CKR ligand, CCL19. 4T1.2 cells expressing CCX-CKR exhibited an enhanced ability to scavenge CCL19 compared with control cells indicating that CCX-CKR is functionally expressed at the protein level by 4TCCX cells (Figure 1a). Before examining the effect of CCX-CKR expression on tumour growth and metastasis in vivo, the transfectants were tested for their growth properties in vitro. Importantly, the data revealed no differences in either anchoragedependent or -independent growth between 4TCCX and 4TpEF cells in vitro (Supplementary Figures 1b, c). The effect of CCX-CKR expression on 4T1.2 tumour progression was next examined. When injected into the mammary fat pad of syngeneic Balb/c mice, the growth of 4TCCX tumours was significantly reduced compared with control 4TpEF tumours (Figures 1b, c). Surprisingly, however, when the lungs of 4TCCX tumour-bearing mice were examined for the extent of spontaneous metastasis, there was a significantly increased number of metastatic surface nodules compared with control tumour-bearing mice (Figures 1d, e). To determine whether the observed increase in metastasis occurred independently of the growth of the primary tumour, experiments were performed to assess the ability of the cells to colonise lungs when they were injected directly into the tail veins of Balb/c mice. The data indicated that 4TCCX cells were able to colonise lungs more efficiently compared with control cells upon intravenous administration (Figures 1f, g). CCX-CKR does not function as a chemokine scavenger on 4T1.2 cells in vivo Given the proposed function of CCX-CKR as a chemokine scavenger, and as 4TCCX cells showed higher CCL19 scavenging ability than control cells in vitro (Figure 1a), the effects of expression of Immunology and Cell Biology

CCX-CKR on chemokine scavenging in vivo when expressed on 4T1.2 tumours were tested. Tumour homogenate supernatants were assayed for levels of CCX-CKR ligands, CCL19, CCL21 and CCL25. Unexpectedly, levels of these chemokines in 4TCCX tumours were not different from the control tumours throughout the course of tumour progression (Figures 2a–c, and Supplementary Figures 2a–c). We have confirmed that this was not due to the loss of CCX-CKR expression by tumour cells over time (Supplementary Figure 2d). The results of these experiments are not consistent with a role for CCX-CKR as a chemokine scavenger on 4T1.2 cells in vivo and suggest that alternative, chemokine-scavenging independent, mechanisms may contribute to the observed effect of CCX-CKR expression on mammary tumour progression and metastasis. As the major biological function of chemokines and chemokine receptors is to orchestrate immune responses through regulation of leukocyte trafficking, we next investigated the requirement for the immune system in the observed effects of CCX-CKR expression in 4T1.2 cells on primary tumour growth and metastasis. The growth of 4TCCX tumours remained inhibited in severe combined immunodeficiency (SCID) mice, which lack functional T and B cells and inhibition remained unchanged when NK cells were depleted from these SCID mice (Figures 2d, e). Furthermore, in these immunodeficient mice, enhanced spontaneous and haematogenous metastasis of 4TCCX tumours to the lungs was also observed (data not shown and Figures 2f, g). Although a role for other innate cellular effectors such as neutrophils and monocyte/macrophages was not investigated, there is presently no evidence that the ligands for CCX-CKR affect these other innate cells to our knowledge. Together, these data suggest that it is unlikely that inhibition of primary 4TCCX tumour growth and enhancement of metastasis are mediated through changes in chemokine ligand levels in the tumour microenvironment or altered anti-tumour immunity. This suggests that expression of CCX-CKR may have effects on other intrinsic characteristics of 4T1.2 cells. Given the observation that expression of CCX-CKR aggravated spontaneous and haematogenous metastasis, it is possible that the expression of CCX-CKR renders the 4T1.2 cells intrinsically more malignant, that is, more motile and invasive, which may cause rapid shedding of cells from the primary tumours resulting in smaller primary tumours with extensive metastatic nodules. Therefore, the effect of CCX-CKR expression on these aspects of tumour cell biology of 4T1.2 cells was next explored. Expression of CCX-CKR enhances metastasis of 4T1.2 cells Tumour progression and metastasis are complex, multi-step processes involving various factors and cellular responses. Direct involvement of CCX-CKR in tumour cell biology is yet to be definitively demonstrated. Therefore, investigations were conducted to determine whether expression of CCX-CKR changes cellular characteristics and responses involved in different stages of tumour progression. The process of spontaneous metastasis first involves detachment of cells from the primary tumour, which then invade through the extracellular matrix (ECM) and intravasate across endothelial barriers into circulation. Subsequently, tumour cells arrest in capillary beds of a secondary site where extravasation into the secondary tissue to form metastases occurs. Our data support the hypothesis that expression of CCX-CKR impacts on the later steps of metastatic process (Figure 1f) but do not exclude the possibility that upstream effects also contribute. Therefore, to test whether expression of CCX-CKR influenced the initial steps of 4T1.2 metastasis, the ability of 4TCCX and 4TpEF cells to escape from the primary tumour, intravasate into and survive in the circulation was assessed in vivo. We observed

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Figure 1 CCX-CKR suppresses primary tumour growth but enhances metastasis. (a) Ability of 4T1.2 cells to scavenge its ligands. Cells were incubated with recombinant mouse CCL19 for 3 h and the remaining CCL19 in the supernatant was measured by ELISA (n ¼ 12). (b and c) In vivo growth of 4T1.2 tumours. Cells were injected into the mammary fat pad of female Balb/c mice and tumours were measured every 2 days (b) and weighed when mice were killed (c). (d) Spontaneous metastasis of 4T1.2 tumours in vivo. When mice were killed lungs were harvested and the number of surface metastatic nodules was counted under a stereomicroscope (n ¼ 40). (e) Representative images of lungs from 4TpEF (left) or 4TCCX (right) tumour-bearing mice. (f) Haematogenous metastasis of 4T1.2 cells. Balb/c mice were injected intravenously with 2  105 cells and the number of lung surface nodules was counted on the day 14 post injection (n ¼ 16). (g) Representative images of lungs from 4TpEF (left) or 4TCCX (right) injected mice. Data are represented as mean±s.e.m., **Po0.01; ***Po0.001.

increased numbers of tumour cells in the circulation of mice-bearing 4TCCX tumours compared with control tumour-bearing mice (Figure 3a). This indicated that the expression of CCX-CKR has a role in one or more of the initial steps of tumour metastasis, including (1) detachment from the primary tumour, (2) invasion, (3) migration and/or (4) survival in the blood circulation. Therefore, the effect of CCX-CKR expression on these aspects of cellular behaviour was examined in vitro. To determine whether CCX-CKR expression alters detachment from primary tumours, ECM adhesion and cell–cell adhesion of 4T1.2 cell lines were examined. 4TCCX cells were less able to adhere to Matrigel compared with control cells (Figure 3b). Furthermore,

when cells were added to wells precoated with a monolayer of the same cell line, 4TCCX cells were less capable of homotypic adhesion compared with control cells (Figure 3c). When the ability of 4T1.2 cells to invade through the basement membrane was tested using Matrigel, significantly higher numbers of 4TCCX cells invaded though Matrigel compared with control cells (Figure 3d). Furthermore, 4TCCX cells also had increased motility compared with control cells (Figure 3e). Together, these data indicate that CCX-CKR inhibits 4T1.2 adhesion to the ECM and to each other, while increasing their motility and ability to invade surrounding tissues. The next key step in the process of metastasis is dissemination through the circulation. Tumour cells often acquire resistance to Immunology and Cell Biology

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Figure 2 The effect of CCX-CKR on tumour progression is independent of the host anti-tumour immune response. (a–c) Levels of CCX-CKR ligands in 4T1.2 tumours in vivo. Tumours harvested on day 28 p.i. were homogenised and supernatants were tested for the levels of CCL19 (a), CCL21 (b) and CCL25 (c) by sequential ELISA (n ¼ 10). (d and e) Growth of 4T1.2 tumour in SCID mice depleted of NK cells. On day 0, either 4TpEF or 4TCCX cells were injected into the mammary fat pad of female SCID mice and on days 1, 0, 7, 14 and 21, mice were injected intraperitoneally with either normal rabbit IgG (NRIgG; isotype control) or anti-asialoGM1 (NK cell-depleting antibody). Tumours were measured every 2 days (d) and weighed when mice were killed (e; n ¼ 5). (f) Haematogenous metastasis of 4T1.2 cells in SCID mice depleted of NK cells. Mice were injected intravenously with 2  105 cells on day 0 and the number of lung surface nodules was counted on the day 14 post injection. On days 1, 0, 7, mice were injected intraperitoneally with either normal rabbit IgG or anti-asialoGM1 (n ¼ 5). (g) Representative images of lungs from 4TpEF (left) or 4TCCX (right) injected mice treated with NRIgG (top) or ani-asialoGM1 (bottom). Data are represented as mean±s.e.m., *Po0.05; **Po0.01; ***Po0.001.

Immunology and Cell Biology

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Figure 3 CCX-CKR enhances tumourigenicity of 4T1.2 cells. (a) Ability of 4T1.2 tumour cells to intravasate and survive in circulation in vivo. Cells were injected into the 4th mammary fat pad of Balb/c mice and on days 14, 21 and 28, blood was collected from right atrium and cells were cultured in the presence of puromycin until colonies were formed (n ¼ 6). (b) ECM adhesion of 4T1.2 cells in vitro. Cells were labelled with Calcein-AM and added to wells coated with Matrigel. After a 30-min incubation at 37 1C, the tray was scanned before and after unbound cells were washed away to determine percentage adhesion (n ¼ 6). (c) Homotypic adhesion of 4T1.2 cells in vitro. Cells were labelled with Calcein-AM and added to wells where a monolayer of cells had been allowed to form overnight. After a 90-min incubation at 37 1C, the tray was scanned before and after bound cells were fixed with 4% formaldehyde solution and unbound cells were washed away to determine percentage adhesion (n ¼ 6). (d) Invasion of 4T1.2 cells through ECM in vitro. Cells were seeded within a Matrigel layer on the upper side of Transwell chambers and incubated for 24 h at 37 1C with or without 1% mouse serum in the lower chambers (n ¼ 9). (e) Migration of 4T1.2 cells in vitro. Cells were added to the upper chambers of blind well with or without 1% mouse serum in the lower chambers. After a 6 h incubation at 37 1C, cells that migrated through the membrane to the lower chambers were transferred to a tissue culture tray and cultured until colonies were formed. Representative images of colonies formed (right; n ¼ 9). Data are represented as mean±s.e.m., *Po0.05; **Po0.01; ***Po0.001.

anoikis (detachment-induced apoptosis), enabling them to survive in circulation after losing attachment to the ECM. Therefore, whether CCX-CKR influenced anoikis was determined by measuring apoptosis of the 4T1.2 cell lines cultured in suspension. Western blot analysis of pro-apoptotic proteins poly (ADP-ribose) polymerase and caspase-3 in cells cultured in suspension in the absence of serum revealed that expression of CCX-CKR inhibited expression of these markers of apoptosis (Figure 4a). Flow cytometric anoikis assays also revealed that expression of CCX-CKR in 4T1.2 cells inhibited anoikis both in the presence and absence of serum (Figures 4b and c). Together, this demonstrates that the expression of CCX-CKR both inhibits apoptosis and increases resistance to anoikis. Expression of transforming growth factor (TGF)-b1 is increased in CCX-CKR-expressing 4T1.2 cells The characteristics displayed by 4TCCX cells are typical of tumour cells that have undergone epithelial–mesenchymal transition (EMT), including loss of adhesion, enhanced motility and invasiveness through ECM and increased resistance to anoikis. Therefore, to assess whether expression of CCX-CKR has accelerated the EMT in 4T1.2 cells, changes in expression of hallmark EMT markers on these cells were examined. Consistent with the observation that homotypic

adhesion of 4TCCX cells was decreased compared with control cells, expression of E-cadherin was found to be significantly reduced in 4TCCX cells at both mRNA and protein levels (Figures 5a, b). Furthermore, vimentin, an important mesenchymal marker that is expressed at low levels in normal epithelial cells, was significantly increased in 4TCCX cells compared with control cells (Figure 5c). The decrease in E-cadherin levels and increase in vimentin levels were also observed in in vivo 4TCCX tumours compared with 4TpEF tumours (Figures 5b, d). Together the data suggest that expression of CCX-CKR enhances tumourigenicity of 4T1.2 cells by accelerating the EMT of these cells. To assess how expression of CCX-CKR may accelerate the EMT in 4T1.2 cells, the effect of CCX-CKR expression on TGF-b1, a potent inducer of EMT,20,21 and its signalling in 4T1.2 cells was examined. The level of TGF-b1 expressed by 4TCCX cells was markedly increased at both mRNA and protein levels compared with control cells (Figures 6a, b). Furthermore, a similar increase in the levels of TGF-b1 was also observed in 4TCCX tumours compared with 4TpEF tumours in vivo (Figures 6c, d). The EMT process is initiated by binding of TGF-b1 to its cognate type II TGF-b receptor (TGF-bRII), which transphosphorylates the type I TGF-b receptor (TGF-bRI) leading to phosphorylation of Smad 2 and Smad 3. Phosphorylated Immunology and Cell Biology

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2/3 phosphorylation were apparent in 4TCCX cells compared with control cells (Figures 6e, f). This increase in phosphorylated Smad 2/3 is not due to an increase in expression of TGF-bRI/II in 4TCCX cells, as our qPCR data showed approximately 20% reduction in mRNA of these genes in 4TCCX cells compared with control cells (data not shown). These data together suggest that expression of CCX-CKR results in induction of TGF-b1 expression in 4T1.2 cells leading to increased autocrine activation of TGF-b1 signalling, thereby accelerating the EMT in 4T1.2 cells.

Figure 4 CCX-CKR increases resistance to anoikis of 4T1.2 cells. (a) Levels of pro-apoptotic proteins in 4T1.2 cells cultured under anoikis-inducing condition. Cells were cultured in Ultra-low attachment or standard tissue culture trays. The pro-apoptotic proteins, poly (ADP-ribose) polymerase (PARP) and caspase-3 were detected by western blot analysis. Representative blot from three independent experiments. (b and c) Frequency of apoptotic 4T1.2 cells cultured under anoikis-inducing conditions. Cells were cultured as described in a and fragmented DNA was extracted from the cells and remaining DNA was stained with PI. Shown are representative histograms from two independent experiments (b) and the frequency of 4T1.2 cells undergone anoikis was estimated by subtracting the percentage of apoptotic cells in adherent condition from suspended cells (c; n ¼ 6). Data are represented as mean±s.e.m., *Po0.05; ***Po0.001.

Smad 2/3 form a heterotrimer with Smad 4, which is translocated to the nucleus to interact with transcription factors, co-activators and co-repressors to suppress epithelial-specific genes and promote expression of mesenchymal-specific genes.22–25 When read-outs of TGF-b1 signalling in 4T1.2 cells were examined, higher levels of Smad Immunology and Cell Biology

DISCUSSION The results of this study provide new insights into the role and function of CCX-CKR when expressed on mammary tumour cells in vivo. We have shown that CCX-CKR expressed on 4T1.2 cells does not appear to function as a chemokine scavenger in vivo and does not regulate the host anti-tumour immune response. Instead, it appears to promote the expression of TGF-b1 and accelerate the EMT of tumours leading to enhanced metastasis. Although we have previously shown that the endogenously expressed CCX-CKR in the mouse functions to regulate bioavailability of its ligands in vivo,18 the data from the current study clearly showed that the exogenously expressed CCX-CKR on mammary tumour cells does not display this function when cells are transplanted in vivo. Although it is possible that a feedback loop exists for production of chemokines, which may have confounded the apparent levels of chemokines in CCX-CKR-overexpressing tumours, it is more likely from the data obtained during the current study that CCX-CKR overexpressed on 4T1.2 cells does not alter chemokine levels in 4T1.2 tumours. Furthermore, previous studies investigating the immune response against the 4T1.2 tumour have demonstrated that NK cells and CD8 þ T cells have significant roles in reducing the progression and metastasis of 4T1.2 tumours.26,27 Therefore, despite the wellcharacterised function of chemokines and chemokine receptors, including CCX-CKR, in the regulation of the immune response,17,18 the observed effect of CCX-CKR expression on 4T1.2 tumour progression and metastasis was shown to be independent of the host immune response, indicating a non-immune mechanism at play. These results suggest that the function of CCX-CKR may be context dependent and give rise to a possibility that CCX-CKR may have a novel function yet to be identified. The key observation in the current study that supports this possibility is that the expression of CCX-CKR led to an increase in TGF-b1 expression by 4T1.2 cells. Although mechanisms by which TGF-b1 transcription is activated are not well documented in the literature, there is evidence suggesting that angiostensin II is capable of inducing expression of TGF-b through its receptor AT1.28,29 AT1 is a G-protein-coupled receptor and like chemokine receptors, AT1 can induce G-protein-mediated signalling and can activate serine threonine kinases including those in the MAP kinase/Erk pathway30– 32. It is believed that the MAP kinase/Erk pathway activates transcription of TGF-b.33 Although CCX-CKR has been shown to be incapable of inducing classical G-protein-mediated signalling pathways upon ligand binding in CCX-CKR-expressing cells15,34 recently, Watts and colleagues demonstrated that CCX-CKR is capable of recruiting b-arrestin and is able to activate the cAMP/ CRE pathway when activity of Gi proteins is inhibited.35 This indicates that the ability of CCX-CKR to induce signalling events may be context-dependent as seen with another chemokine-binding protein, CXCR7. Indeed, although CXCR7 was originally reported to be incapable of inducing downstream signalling upon ligand binding in various types of cells,36–38 in different systems, activation of

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Figure 5 CCX-CKR accelerates EMT in 4T1.2 cells and in vivo tumours. (a and b) Expression of E-cadherin by 4T1.2 cells and tumours detected by qPCR (a; n ¼ 9) and western blot analysis (b). Representative blot from three independent experiments. (c and d) Expression of vimentin by 4T1.2 cells (c; n ¼ 9) and tumours (d; n ¼ 6) detected by qPCR. Data are represented as mean±s.e.m., *Po0.05; **Po0.01; ***Po0.001.

downstream signalling pathways has been detected, including phosphorylation of Akt in a prostate cancer cell line,39 MAPK activation in HEK293 cells and migration of vascular smooth muscle cells.40 Given the limited reports on intracellular biochemistry of CCX-CKR, and hence, limited understanding of downstream effects of this receptor, it is possible that alternative nonG-protein pathways, such as PI3K/Akt or MAPK/Erk, are activated downstream of CCX-CKR in 4T1.2 cells, and that increased activation of those pathways leads to increased expression of TGF-b1. In this regard, it is also possible that CCX-CKR contributes to an as yet unknown heterodimeric or multimeric receptor, which regulates TGFb1 expression. The ability of G-protein-coupled receptors to form multimeric complexes, principally heterodimers with other G-protein-coupled receptors and other types of receptors, including receptor tyrosine kinases, as we have previously shown for insulin-like growth factor-1 receptor,41 is becoming increasingly well documented. Such receptors represent novel forms of posttranslational regulation of a number of biologically important proteins to form novel receptors with novel signalling capabilities. Another insight from our study is that expression of CCX-CKR inhibited primary tumour growth while enhancing metastasis. Although this may be due to enhanced EMT in these cells resulting in rapid shedding from primary tumours as mentioned above, it may also be due to the increased autocrine signalling of TGF-b1. It is now a well-established concept that TGF-b has opposing effects on tumour progression that are context- and stage-dependent.42,43 There are a number of pieces of evidence indicating that at early stage of tumourigenesis TGF-b can inhibit proliferation44,45 and induce cell

apoptosis46,47 or cell senescence,48,49 including in the mammary stem cell population.50 However, it has been shown in various types of tumours including mammary tumours that accumulation of genetic alterations as tumours progress leads to attenuation of the growth inhibitory effect of TGF-b and increases in TGF-b can drive malignant progression and metastasis through induction of EMT.51– 53 As we have shown that the expression of CCX-CKR results in induction of TGF-b1, it appears that the autocrine effect of TGF-b1 on 4T1.2 cells at the initial stages of tumour growth may be tumour suppressive, but as the tumour progresses the effect becomes protumourigenic rendering the cells less adherent, more motile and invasive as well as resistant to anoikis. In summary, the findings of this study have provided evidence that the atypical chemokine receptor CCX-CKR has a novel function as a regulator of TGF-b1 expression in 4T1.2 mammary carcinoma cells. Expression of CCX-CKR inhibits primary tumour growth but enhances metastasis apparently independently of its known function as a chemokine scavenger or through its effects on the immune response. We attribute these changes to increased TGF-b1 production induced by CCX-CKR that operates in an autocrine manner to promote the EMT in mammary carcinoma cells. To fully understand how CCX-CKR activates TGF-b1 expression in these cells, it is essential that a better understanding of the behaviour of CCX-CKR on the cell surface and the intracellular events downstream of CCX-CKR potentially triggered in 4T1.2 cells is attained. Nevertheless, our data indicate that CCX-CKR has a significant role in TGF-brelated tumour biology and therefore support the contention that a better understanding of the interplay between CCX-CKR Immunology and Cell Biology

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Figure 6 CCX-CKR enhances autocrine signalling of TGF-b in 4T1.2 cells. (a and b) Expression of TGF-b1 by 4T1.2 cells detected by qPCR (a) and ELISA (b; n ¼ 9). (c and d) Expression of TGF-b1 by in vivo tumours detected by qPCR (c) and ELISA (d; n ¼ 6). (e and f) autocrine phosphorylation of Smad 2/3 in 4T1.2 cells. Cells were stimulated for 3 min and intracellular phospho-Smad 2/3 was detected in flow cytometric analysis. (c) Mean fluorescent intensity (MFI) of anti-phospho-Smad 2/3 antibody-stained cells (n ¼ 9). (d) Representative histograms for isotype control antibody- and anti-phospho-Smad 2/3 antibody-stained cells. Data are represented as mean±s.e.m., *Po0.05; **Po0.01; ****Po0.0001.

and TGF-b pathways may lead to new avenues investigating the pathophysiology of breast cancer and of other TGF-b-related diseases. MATERIALS AND METHODS Cell lines 4T1.2 cells were donated by R. Anderson54 (Peter MacCallum Cancer Centre, Melbourne, Australia). Cells were maintained in minimum essential medium alpha (aMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 0.2 U ml 1 penicillin/gentamycin at 37 1C in 5% CO2. Cells were transfected with pEF-IRES-puro6 (pEF) with or without the coding sequence of murine CCX-CKR insert using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Transfectants were selected with 3 mg ml 1 puromycin (Millipore, Billerica, MA, USA).

Mice Female Balb/c mice and SCID mice were purchased from the Animal Resource Centre (Perth, WA, Australia) or University of Adelaide Waite campus Laboratory Animal Services (Adelaide, SA, Australia). SCID mice were kept in pathogen-free conditions. All experimental protocols used in this study were approved by the University of Adelaide Animal Ethics Committee.

qPCR Total RNA was extracted using TRI Reagent (Life Technologies) according to the manufacturer’s instructions. Purified RNA was further clarified in 2 M NaCl and 100% ethanol, then treated with TURBO DNA-free (Life Technologies) according to the manufacturer’s instructions. cDNA synthesis was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Basel, Switzerland) with or without reverse transcriptase according to the manufacturer’s instructions. qPCR reactions were performed using SYBR Green I master mix (Roche Applied Science) according to the manufacturer’s instructions and as Immunology and Cell Biology

previously described.18 Primers for CCX-CKR were 50 -AGATGCAGC AGATCCGCAT-30 (forward) and 50 -CAGTGAGCTTCCCGTTCAG-30 (reverse), for E-cadherin 50 -CAGGTCTCCTCATGGCTTTGC-30 (forward) and 50 -CTTCCGAAAAGAAGGCTGTCC-30 (reverse), for vimentin 50 -CTTG AACGGAAAGTGGAATCCT-30 (forward) and 50 -GTCAGGCTTGGAAACG TCC-30 (reverse), for TGF-b1 50 -GAGGTCACCCGCGTGCTA-30 (forward) and 50 -TGTGTGAGATGTCTTTGGTTTTCTC-30 (reverse) and for RPLP0 50 AGATGCAGCAGATCCGCAT-30 (forward) and 50 -CAGTGAGCTTCCCG TTCAG-30 (reverse).

Scavenging assay Cells (2  105) were incubated in medium containing either 5 or 10 ng ml 1 of recombinant mouse CCL19 (R&D Systems, Minneapolis, MN, USA) at 37 1C for 3 h during which time the cells were resuspended by inversion of the tubes every 30 min. After the incubation, 100 ml of the supernatants were collected for detection of remaining CCL19 by ELISA as described previously.18

In vitro anchorage-dependent and -independent growth For anchorage-dependent growth, cells (2.5  103) were seeded in 24-well trays. On days 0, 2, 5 and 7, the rate of proliferation was measured using sodium 30 -[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6nitro) benzene sulfonic acid hydrate (1 mg ml 1)/N-methyl dibenzopyrazine methyl sulphate (1.25 mM) assay (combined at 50:1). For anchorage-independent growth, the soft agar assay was performed as described previously.26

In vivo primary tumour growth and spontaneous metastasis Female Balb/c mice or SCID mice aged between 6 and 8 weeks old were injected with 1  105 cells in endotoxin-free phosphate-buffered saline (PBS) into a mammary fat pad. From day 7 post injection, the perpendicular diameters of each tumour were measured using digital callipers and the tumour sizes were calculated as multiples of shortest and longest diameters.

Novel function of CCX-CKR in mammary carcinoma Y Harata-Lee et al 823 Mice were killed once the tumour diameter reached 15 mm or became ulcerated and tumours were removed and weighed. When the mice were killed, lungs were also harvested and fixed in 4% formaldehyde solution overnight and the number of metastatic nodules was counted under a stereomicroscope and the images of the lung lobes were captured on a Leica MZ16FA Stereomicroscope (Leica Microsystems, Wetzlar, Germany). For NK cell depletion experiments, mice were injected intraperitoneally with 500 mg of normal rabbit IgG or anti-asialoGM1 (Wako, Osaka, Japan) on days 1, 0 and every 7 days following tumour injection.

Haematogenous metastasis Mice were injected with 2  105 cells in endotoxin-free PBS intravenously into the tail vein. On day 14 post injection, the lungs were removed and fixed in 4% formaldehyde solution overnight and the number of metastatic nodules was counted under a stereomicroscope and the images of the lung lobes were captured on a Leica MZ16FA Stereomicroscope.

Sequential ELISA for chemokines Sequential ELISAs for chemokines were performed in the order of CCL19, CCL21 and CCL25. Tumours were mechanically homogenised in PBScontaining protease inhibitors (Sigma-Aldrich, St Louis, MO, USA, USAMO). The EIA/RIA high binding 96-well plates (Corning Life Science, Corning, NY, USA) were coated with capture antibodies (CCL19: 2 mg ml 1, CCL21: 500 ng ml 1, CCL25: 2 mg ml 1; R&D Systems, MN, USA) prepared in 100 mM NaHCO3 coating buffer. All the subsequent steps were performed on one plate at a time in sequence. First, the wells were blocked with 5% skim milk before recombinant proteins and sample supernatants were added. For standard curves, recombinant proteins (CCL19: 300 ng ml 1, CCL21: 750 ng ml 1, CCL25: 1 mg ml 1; R&D Systems) were prepared together in PBS containing 0.5% skim milk, 0.1% bovine serum albumin (BSA) and 0.005% Tween-20 and serially diluted 1:2 in U-bottom 96-well plate. The recombinant protein mix and sample supernatants in the first plate were sequentially transferred from the first plate to the next. The plates were then incubated with biotin-conjugated antibodies (CCL19: 200 ng ml 1, CCL21: 50 ng ml 1, CCL25: 200 ng ml 1; R&D Systems) followed by strep-HRP (1:10 000) prepared in PBS containing 0.1% BSA and 0.005% Tween-20. 3,30 ,5,50 -Tetramethylbenzidine substrate solution (eBioscience, San Diego, CA, USA) were then added to the wells and incubated in dark until the appropriate level of colour developed. Finally, 3 M HCl solution was added to stop the reaction. Between each step wells were washed with PBS containing 0.05% Tween-20 three times. The absorbance was measured at 450 nm using Biotrak II Plate reader (GE Healthcare Life Science, Pittsburgh, PA, USA).

In vivo intravasation assay Mice were injected with 4T1.2 cells into the mammary fat pad as described above. On days 14, 21 and 28 post injection, mice were killed and blood was collected from the right atrium. Erythrocytes were removed and the remaining cells were cultured in a six-well tray in the presence of puromycin until colonies were formed.

unbound cells were washed away. The percentage adhesion was calculated as (fluorescence intensity after washes/fluorescent intensity before washes)  100.

Invasion assay Cells (5  105) were combined with neat Matrigel at 1:1 ratio and the upper side of Transwell membranes (8 mm pores; Corning Life Science) were coated with the cell/Matrigel mixture. The lower chambers were filled with aMEM with or without 1% mouse serum and trays were incubated for 24 h. Cells on the upper side of the membranes were scraped off and the cells on the under side of the membranes were fixed with 100% ethanol and stained with 2% Toluidine blue (Sigma-Aldrich). The number of cells on the bottom side of membranes as well as at the bottom of lower chambers was estimated by counting the number of cells in five random fields. The percentage of invaded cells was calculated as (no. of invaded cells/no. of input cells)  100.

Migration assay The lower chambers of blind well chambers (Neuro Probe, Gaithersburg, MD, USA) were filled with aMEM with or without 1% mouse serum. Cells (5  104) were prepared in aMEM containing 0.1% BSA and added to the upper chamber separated with polycarbonate membranes (8 mm pores; Neuro Probe). After a 6-h incubation, the cells migrated to the lower chambers were transferred to a 12-well tray and cultured until colonies formed. The number of colonies in each well was determined using the Quantity One Version 4.3.1 (Bio-Rad, Hercules, CA, USA). The percentage of migrated cells was estimated as (no. of migrated cells/no. of input cells)  100.

Anoikis assay Cells (2  105) were cultured in either Ultra-low attachment (Corning Life Science) or standard tissue culture six-well trays with or without 5% FBS for 24 h. To measure apoptosis, the cells were subjected to western blot analysis on lysates as well as cell-cycle analysis as described previously.55

Western blot for detection of E-cadherin Twenty micrograms of total protein from either cell lysate or tumour lysate were separated on a Tris NuPage BOLT gel (Life Technologies). E-cadherin was detected using anti-E-cadherin (1:1000; 24E10: Cell Signalling Technologies, Danvers, MA, USA) and anti-rabbit IgG-HRP (1:100 000; Sigma-Aldrich). Equal protein loading was confirmed by detection of b-actin (1:5000; Sigma-Aldrich) and anti-mouse IgG-HRP (1:20 000; Thermo Scientific, Waltham, MA, USA).

Analysis of Smad 2/3 phosphorylation Cells were serum-starved overnight and the medium was replaced with fresh medium. After a 30-min incubation, cells were stained with LIVE/DEAD Near Infrared Fixable Dead Cell Stain (Life Technologies) and permeabilised using Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturers’ instructions. Cells were then stained with either anti-phospho-Smad 2/3 (1:400; Cell Signaling) or rabbit IgG (1:5400; Cell Signaling) followed by anti-rabbit Alexa Fluor 488 (1:200, Life technologies). The stained cells were acquired on an LSRII (BD Biosciences) and the data were analysed using FlowJo software (TreeStar Inc., Ashland, OR, USA).

ECM adhesion assay A 96-well tray was coated with either 0.5 mg ml 1 or 0.1 mg ml 1 Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The wells were then blocked with 1% BSA before 2  104 cells labelled with Calcein-AM (Life Technologies) were added to each well in the presence of 10% FBS. After a 30-min incubation at 37 1C, the tray was scanned before and after unbound cells were washed away. The percentage adhesion was calculated as (fluorescence intensity after washes/ fluorescent intensity before washes)  100.

Statistical analysis All statistical tests were performed using GraphPad Prism version 5.0 for Windows, GraphPad Software (La Jolla, CA, USA, http://www.graphpad.com). Unless otherwise stated, statistical test used for analysis was an unpaired twotailed t-test.

CONFLICT OF INTEREST The authors declare no conflict of interest.

Homotypic adhesion assay Cells (3  104) were cultured in a 96-well tray overnight to form a monolayer. The wells were blocked with 1% BSA and freshly collected cells (either 1  104 or 2  104) labelled with Calcein-AM were added to appropriate wells in the presence of 10% FBS. After a 90-min incubation at 37 1C, the tray was scanned before and after bound cells were fixed with 4% formaldehyde solution and

ACKNOWLEDGEMENTS This study was supported by grants from the National Health and Medical Research Council of Australia. MJS was supported by a NH&MRC Australia Fellowship. We thank Associate Professor R Anderson for provision of 4T1.2 cells. Immunology and Cell Biology

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