Figures
Abstract
Background
S1P3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtype-selective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist.
Methodology/Principal Findings
Specific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3-mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts.
Conclusions/Significance
We have developed the first-reported monoclonal antibody that selectively recognizes a lipid-activated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsis and breast cancer, it also provides proof of concept for the generation of novel GPCR-specific therapeutic antibodies.
Citation: Harris GL, Creason MB, Brulte GB, Herr DR (2012) In Vitro and In Vivo Antagonism of a G Protein-Coupled Receptor (S1P3) with a Novel Blocking Monoclonal Antibody. PLoS ONE 7(4): e35129. https://doi.org/10.1371/journal.pone.0035129
Editor: Irina Agoulnik, Florida International University, United States of America
Received: January 13, 2012; Accepted: March 13, 2012; Published: April 5, 2012
Copyright: © 2012 Harris et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health (NIH) grants (1R43 CA132400, 1R43 CA156892) to DRH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
Competing interests: DRH and MBC were Expression Drug Designs employees during the performance of these studies. DRH, GLH, and GBB own shares in Expression Drug Designs.This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
Introduction
The use of monoclonal antibodies (mAbs) to antagonize transembrane receptors has met with tremendous clinical and commercial success over the course of the past decade. The success of antibody drugs is based on their exquisite specificity and affinity, which are essential components of targeted molecular therapy. With 23 antibody drugs currently approved for clinical use and annual sales in the tens of billions of dollars [1], these biologics are being used for a wide range of indications such as inflammatory diseases, autoimmune diseases, stroke, and heart disease, but the greatest therapeutic antibody success stories involve the treatment of cancer. Examples of some the most effective and widely used, anti-cancer therapeutic antibody drugs include trastuzumab (Herceptin®, a HER2 inhibitor), bevacizumab (Avastin®, a VEGF inhibitor), and panitumumab (Vectibix™, an EGFR inhibitor).
Sphingosine 1-phosphate (S1P) is a lipid signaling molecule (Figure 1) that is present in serum at biologically relevant concentrations (high nanomolar range). S1P is generated by the phosphorylation of sphingosine by sphingosine kinase in the final step of a highly conserved metabolic pathway [2]. Although there have been reports of some intracellular roles of S1P [3]–[5], the majority of its effects are mediated by a family of five known S1P-selective G protein-coupled receptors (GPCRs). These receptors belong to a GPCR subfamily (formerly known as the “Edg” receptors) whose members are activated by S1P (S1P1–5) or the structurally similar lipid, lysophosphatidic acid (LPA; LPA1–3). They couple to a number of G proteins and downstream effectors to elicit a variety of responses in almost every known cell type. The responses vary among cell types depending on the expression profile of the receptors and effectors, but notably include proliferation, survival, and cytoskeletal rearrangement (reviewed in: [6]–[9]).
S1P is an extracellular signaling molecule, generated by the phosphorylation of sphingosine, that exerts a variety of effects on a family of 5 cognate GPCRs.
Previous studies are consistent with a pro-tumorigenic role of S1P. S1P is known to increase the proliferation, survival, motility, and invasiveness of breast tumor cells [10]–[13]. In addition, the known involvement of S1P signaling in the processes of angiogenesis and vascular maturation underscores the importance of this pathway in cancer progression [14], [15]. The tumor-promoting effect of S1P is directly supported by the observation that overexpression of sphingosine kinase in MCF-7 cells promotes tumorigenesis and tumor vascularization in a nude mouse model [10]. Furthermore, it has been demonstrated that neutralization of S1P has a potent tumor-suppressive effect [16], an approach that is currently under clinical investigation.
In breast cancer cells (BCCs) the tumorigenic effects of S1P are likely to be largely mediated by the activation of cognate receptor subtype S1P3. S1P3 is the most highly expressed S1P receptor in BCCs [17], [18], is known to promote cell migration [19]–[21] and EGF responsivity [11], [12], [18], [22], [23], and may mediate the proliferative effects of estrogen [24]. In addition to the direct effects that S1P3 have on BCCs, S1P3 also mediates angiogenesis [25]–[31], thus promoting tumor growth by increasing vascularity. A recent study provided evidence for the clinical importance of S1P3 by showing that expression of S1P3 in breast tumors positively correlates with decreased tamoxifen sensitivity and decreased patient survival [32].
In addition to promoting tumor development, activation of S1P3 is also involved in the pathology of inflammatory responses. This is most clearly illustrated by the fact that mice specifically lacking S1P3 are resistant to the effects of the bacterial endotoxin lipopolysaccharide (LPS) [33]. There is near complete attenuation of inflammatory cytokine release in S1P3-/- mice following LPS challenge. Most importantly, when LPS is administered at a dose that is lethal to 90% of wild-type mice, more than 80% of S1P3 knockouts survive. The protective effect of S1P3 loss-of-function is likely due to the roles of S1P3 in both immune cells and endothelial cells. S1P3 has been shown to mediate pro-inflammatory responses in a number of pathological conditions [34]. This is mediated by multiple immune cell types including dendritic cells [35], [36]. Moreover, it is known that activation of S1P3 on endothelial cells causes the disruption of tight junctions and an increase in vascular permeability that results in hemodynamic instability during septic shock [37]–[40]. This pathological activation of S1P3 occurs downstream of thrombin-induced innate immune responses to cause an amplification of cytokine release and inflammation and a loss of vascular integrity [33], [41].
Considering these known pathological roles, we reasoned that specific antagonism of S1P3 would be likely to provide effective protection against breast tumor development and acute systemic inflammation. Since the structural similarities among the “Edg” family of receptors provide significant challenges to the generation of subtype-selective small molecule inhibitors, we elected to pursue the development of a blocking monoclonal antibody. While this approach itself has unique challenges for GPCR targets, we have developed one S1P3-blocking mAb: 7H9. Here we demonstrate the specificity and efficacy of 7H9 and evaluate its therapeutic potential in animal models for sepsis and breast cancer.
Results
7H9 Specifically Binds S1P3
Monoclonal antibody 7H9 was evaluated for specificity to S1P3 by immunocytochemistry (Figure 2). S1P3 is a member of the highly conserved “Edg” family of GPCRs. HEK293 cells were transfected with expression constructs for each of 7 different “Edg” family receptors, then fixed and incubated with purified 7H9 antibody (1 µg/ml). Specific binding to S1P3-expressing cells was apparent by epifluorescence, while binding to the remaining “Edg” receptors was undetectable.
HEK293 cells were transfected with expression constructs for all closely related “Edg” family GPCRs (green), fixed, incubated with purified 7H9 (1 µg/ml), and visualized with a Cy3-conjugated secondary antibody (red). 7H9 specifically recognizes cells expressing S1P3, but not its closest homologs.
7H9 Blocks Activation of S1P3
Since stimulation of S1P3 results in the activation of multiple downstream processes, we evaluated 7H9 for its ability to inhibit a number of distinct S1P3-mediated events.
Arrestin translocation.
The signal amplitude of canonical GPCRs is limited by the arrestin family of regulatory proteins. Upon activation of GPCRs, cytosolic arrestin rapidly binds the receptor, stimulating receptor phosphorylation and subsequent clathrin-dependent internalization of the protein complex into intracellular endosomal vesicles. Vesicular localization of arrestin is indicative of GPCR activation. This can be visualized with the use of fluorescently labeled arrestin constructs [42]. To determine if 7H9 could inhibit S1P3-mediated arrestin translocation, we co-transfected HEK293 cells with epitope-tagged S1P3 and β-arrestin-EGFP (Figure 3A-D). EGFP labeling was diffuse throughout the cytoplasm of unstimulated cells (Figure 3A), but accumulated into distinct puncta 5 minutes after stimulation with 1µM S1P (Figure 3B). This subcellular redistribution was almost completely blocked by pre-incubation of cells with 7H9 (Figure 3C-D).
(A-D) 7H9 blocks S1P3-mediated arrestin translocation. β-arrestin (green) is cytosolic in quiescent cells and appears as diffuse labeling (A). Following stimulation with 1 µM S1P (B), arrestin translocates to the plasma membrane and is rapidly internalized into intracellular vesicles (arrows). In contrast, when cells are pre-treated with 1 µg/ml 7H9 arrestin localization is diffuse and cytoplasmic in both the absence (C) and presence (D) of 1 µM S1P. (E-H) 7H9 blocks S1P-dependent internalization of S1P3. Epitope-tagged S1P3 is normally abundant on the plasma membrane (E, arrows), but is internalized into intracellular vesicles (arrowheads) upon stimulation with 1 µM S1P (F). Following pre-treatment of cells with 7H9, S1P3 remains localized to the plasma membrane in the absence (G) or presence (H) of 1 µM S1P. (I) 7H9 blocks S1P3-dependent calcium mobilization. S1P3-expressing cells exhibited increased intracellular [Ca2+] upon stimulation with 100 nM S1P (blue, antibody control). Cells within the same culture that did not express S1P3-EGFP (green, receptor control) showed no change in [Cai2+]. Similarly, cells expressing S1P3 that were pre-treated with 7H9 (red, 7H9) also showed no response to 100 nM and 1 µM S1P, but gave a partial response at 5 µM. (J) 7H9 blocks S1P3-dependent inhibition of AC. cAMP was measured in S1P3-expressing cells by ELISA and normalized to controls. The graph represents the change in cAMP content from unstimulated cells, relative to the change observed in stimulated cells with no 7H9 pre-treatment. *p<0.05, **p<0.01. Error bars = S.E.M.
Receptor internalization.
S1P3, like most canonical GPCRs, is rapidly internalized into cytoplasmic vesicles upon activation [43]. We investigated whether ligand-dependent internalization of S1P3 could be blocked by 7H9 (Figure 3E-H). RH7777 cells were transfected with epitope-tagged S1P3 constructs, stimulated with 1µM S1P for 15 minutes, fixed, and evaluated by immunocytochemistry. In unstimulated cells, S1P3 is largely localized to the plasma membrane (Figure 3E). After exposure to S1P, labeling becomes punctate and accumulates in the cytoplasm (Figure 3F). This process is dramatically reduced by pre-treatment with 7H9 (Figure 3G-H).
Calcium mobilization.
S1P3 is a Gαq-coupled GPCR and, upon stimulation with S1P, causes an increase in cytoplasmic Ca2+ concentration due to a release of intracellular Ca2+ stores [44], [45]. We investigated whether S1P3-dependent calcium release could be blocked by 7H9 (Figure 3I). RH7777 cells were transfected with S1P3-EGFP fusion constructs, stimulated with S1P, and evaluated for [Cai2+] with calcium indicator dye fura-2. S1P3-expressing cells demonstrated a robust increase in [Cai2+] upon exposure to 100 nM S1P, while S1P3-negative cells in the same culture showed no response. When cells were pre-incubated with conditioned media from 7H9 hybridoma cultures, S1P was unable to elicit the release of Cai2+ until very high, non-physiological concentrations were applied.
Adenylate cyclase (AC) inhibition.
S1P3 also couples to Gαi, and consequently causes the inhibition of AC [45], [46]. This results in a reduction of intracellular cyclic adenosine monophosphate (cAMP). We investigated whether 7H9 could block S1P3-mediated Gαi signaling by measuring S1P-dependent changes in intracellular cAMP (Figure 3J). Stimulation of S1P3-expressing cells with 100 nM S1P resulted in a 35% decrease in intracellular [cAMP]. This effect was partially inhibited by pre-treatment with low concentrations of 7H9, and completely blocked at 10 µg/ml.
7H9 Ameliorates Systemic Inflammation In Vivo
Recent studies in mice demonstrated that deletion of the gene encoding S1P3 conferred resistance to the systemic inflammatory response induced by LPS, and afforded marked protection against the resulting lethality characteristic of disseminated intravascular coagulation (DIC) [33], [41]. We expected that similar protection would be obtained by antagonizing S1P3 in vivo with the administration of 7H9 (Figure 4). Mice were pre-treated with vehicle, non-specific mouse IgG, or 7H9; then were treated with LPS at the reported LD90 dose [33]. Consistent with previous studies, 9 out of 10 mice (90%) died within 4 days of LPS administration in both control groups. However, mice pre-treated with 7H9 were significantly protected, with only 2 of 10 mice (20%) succumbing to systemic inflammation.
Survival curve of mice treated with LPS (8 mg/kg, i.p.) on day 0. Mice were pre-treated with vehicle (PBS), IgG (normal mouse IgG), or 7H9.
7H9 Inhibits the Development of Breast Tumor Xenografts
Since activation of S1P3 is known to contribute to the development and aggressiveness of breast tumors, we investigated whether antagonism of S1P3 with 7H9 could inhibit the progression of human breast tumor xenografts in mice (Figure 5). Solid tumors were established by subcutaneous implantation of MCF7 cells in Foxn1Nu/Nu mice using standard techniques [47]–[49]. When tumors reached ∼200 mm3 by caliper measurement, mice were randomized into 2 groups and treated with normal mouse IgG or 7H9 (1 mg/kg, i.p., QOD), resulting in the stable accumulation of 7H9 in the serum at concentrations that were effective in vitro (Figure S1). After approximately 3 weeks of treatment, tumors in the 7H9 treatment group, on average, demonstrated a detectable decrease in the rate of development relative to the normal IgG control group (Figure 5A). This difference continued to broaden over the next few weeks but fell short of statistical significance (p = 0.16) when the experiment was terminated on day 50. Interestingly, however, upon histological examination we noticed a visible increase in the “necrotic” regions of the 7H9-treated tumors (Figure 5B-C). Quantification revealed that this was a statistically significant, 4-fold increase (Figure 5D).
(A) Volumes of subcutaneous MCF7 tumors in nude mice were determined by caliper measurement. (B-C) Photomicrographs showing H&E stained tumors from IgG control (B) and 7H9-treated (C) mice. (D) Volumes of “necrotic” regions were determined by measuring their cross-sectional areas relative to total area of section. *p<0.05.
Discussion
While the use of therapeutic mAbs has achieved remarkable clinical success, the application of this technology to GPCR drug targets has been lagging, due primarily to the difficulty in generating selective antibodies for critical motifs in these complex, membrane spanning proteins. This is particularly problematic for the many GPCRs with lipid ligands, which typically have inaccessible transmembrane binding pockets. Here, we describe the generation of the first-reported mAb that selectively binds a lipid-activated GPCR, and blocks functional activity.
The strategy underlying the design of 7H9 involved the generation of an antibody that would bind an extracellular loop that formed the opening of the ligand binding pocket, thus preventing ligand-receptor interaction by steric hindrance. This is consistent with the results of the calcium assay (Figure 3I) in which normal, physiological concentrations of S1P had no effect, but unnaturally high concentrations of S1P (∼10-fold higher than normal serum levels) could overcome the inhibition. While this likely plays a role in the blocking effect of 7H9, the results of the functional assays suggest that the mechanism of action is more complex. Specifically, 7H9 at a concentration of 1 µg/ml (∼7 nM) provided efficient antagonism in arrestin, internalization, and calcium assays (Figure 3A-I), but a higher concentration was required for complete inhibition of the cAMP response (Figure 3J). While this may simply be the result of variation between experimental systems, it is possible that 7H9 acts as a somewhat biased antagonist, for example, by altering the confirmation of S1P3 in a way that decreases its affinity for Gαq to a greater extent than that of Gαi. Additional functional studies with greater quantitative resolution have been planned to distinguish between these possibilities.
While the functional studies provide an initial demonstration of S1P3 antagonism, the best evidence for efficacy was provided by the in vivo studies. Notably, administration of 7H9 faithfully phenocopied the genetic null S1pr3-/- mouse in the LPS challenge, using the discrete and unambiguous readout of animal survival (Figure 4). While the effect of 7H9 in the xenografts was less discrete, there was a trend toward inhibition of tumor growth with 7H9 administration (Figure 5A). The fact that this did not reach statistical significance may be due to a number of factors. We expect the direct effect of S1P3 antagonism to be growth-inhibitory rather than cytotoxic, therefore, the timeframe of this model may be too short before significance was reached. After 50 days, the tumors reached a mass that necessitated euthanasia. Alternatively, the extent of the tumor-suppressive effect of 7H9 may not have been completely represented by measurement of tumor volume alone. Indeed, this is supported by the histological analysis of the tumors (Figure 5B-D). As was previously reported, disruption of S1P signaling causes an increase in necrotic lesions in tumor xenografts [16], likely due to decreased tumor angiogenesis.
The recent FDA approval of an S1P receptor-modulating drug for the treatment of multiple sclerosis [50] has brought a great deal of attention to this sub-class of GPCRs in the context of drug development. Significant evidence supports the idea that specific antagonism of S1P3 should be highly effective in treating a number of diseases including sepsis [33], [41], and breast cancer [32]. The interaction between S1P3 and estrogen signaling suggests that such a treatment would be particularly effective as co-therapy with tamoxifen for the treatment of breast cancer [32], [51]. This is consistent with our preliminary observations and is the subject of ongoing study. Our initial pilot study shows that 7H9 may enhance the effect of tamoxifen treatment (Figure S2). While there was no additive effect of combined 7H9/tamoxifen treatment on tumor volume (Figure S2A), the addition of 7H9 resulted in a further decrease in the number of mitotic cells in the tumor relative to tamoxifen alone (Figure S2B). Furthermore, there is also a trend toward increased tumor necrosis with combination therapy (Figure S2C). The fact that this parameter did not reach statistical significance is likely due to the small cohort size (N = 3) and awaits validation.
Since 7H9 is the only known high-affinity, S1P3-selective antagonist, this mAb is a viable candidate for development as a lead drug compound. It is also unlikely to be associated with significant adverse side effects. This is based on 3 lines of evidence: 1) Mice treated with 7H9 in this study exhibited no gross signs of toxicity, 2) S1P3 knockout mice are phenotypically indistinguishable from wild-type littermates [46], and 3) FTY720, a functional antagonist for multiple S1P receptors including S1P3, is well-tolerated in humans [52], [53]. Interestingly, administration of FTY720 has been shown to inhibit tumor growth and angiogenesis in vivo [54], [55], however, this compound acts as a potent immunomodulator through its action on S1P1. While this effect contributes to its efficacy in the treatment of multiple sclerosis, it is highly undesirable in cancer patients for which intact immune surveillance acts to prevent the development of metastatic lesions.
The identification of 7H9 demonstrates that we have developed a technology that is capable of generating specific blocking antibodies for an important but problematic class of GPCR drug targets. We are currently pursuing the development of mAbs for additional lipid-activated GPCRs to show that this approach may be generalized to multiple high-value targets.
Materials and Methods
Ethics Statement
Animal protocols were approved by the Institutional Animal Care and Use Committee at San Diego State University (protocol #: 10-01-00H and 11-02-003H) and conform to National Institutes of Health guidelines and public law. All efforts were made to minimize suffering.
Reagents
Sphingosine-1-phosphate, D-erythro (S1P, BML-SL140-0001, Enzo Life Sciences, Inc, Farmingdale, NY, USA) was maintained as a 1mM stock solution in methanol and stabilized by diluting 1∶10 with 10% fatty acid-free bovine serum albumin (BSA) before making final working concentrations by diluting with Dulbecco’s modified Eagle medium (DMEM).
Generation of 7H9
S1P3-blocking antibody 7H9 was generated by immunizing mice with a synthetic peptide (KKTFSLSPTVWFLREG) and generating hybridoma clones using standard protocols. Clones were selected with a proprietary screening platform. Out of 2,592 candidates, one clone (7H9) demonstrated suitable S1P3-specific binding. Unless otherwise indicated, all studies were performed using antibody that was purified from ascites fluid by protein A/G affinity chromatography (Thermo Fisher Scientific, Rockford, IL, USA).
Cell Lines and Tissue Culture
HEK293 (CRL-1573), RH7777 (CRL-1601), C6 (CCL-107), and MCF7 (HTB-22) cells were obtained from American Type Culture Collection (ATCC). All cell lines were grown as monolayers in humidified incubators at 37°C with 5% CO2. Cells were cultured in DMEM, 25mM D-glucose, 4mM L-glutamine, 1 u/ml penicillin, 1 µg/ml streptomycin, 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).
Immunocytochemistry
For all studies involving immunolabeling, cells were cultured on collagen-coated coverslips (cat #08-115, Millipore, Billerica, MA, USA), fixed with 4% paraformaldehyde, incubated overnight with mouse monoclonal anti-V5 (cat #R960-25, Invitrogen, Carlsbad, CA, USA) diluted 1∶500 in blocking solution (phosphate buffered saline (PBS)/2.5% BSA/0.3% Triton X-100), then incubated with Cy3-anti-mouse (cat #AP192C, Millipore, Billerica, MA, USA) diluted to 1 µg/ml in blocking solution.
Arrestin Translocation
Enhanced green fluorescent protein (EGFP) was cloned in frame to the C-terminus of full length human β-arrestin in vector pcDNA3.1 to generate a β-arrestin-EGFP fusion protein expression construct. Epitope-tagged S1P3 was generated by amplification of S1P3 from cDNA derived from MCF7 breast cancer cells (cat #HTB-22, ATCC) by polymerase chain reaction, and cloning into pcDNA3.1?V5-His TOPO (cat #K4800-01, Invitrogen, Carlsbad, CA, USA) per protocol. These constructs were co-transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) per protocol. Cells were incubated in serum-free media 4 to 16 hours prior to stimulation with S1P. Cells were treated with 1mM S1P or BSA for 15 minutes, fixed and processed for immunocytochemistry as described above, and visualized by fluorescence microscopy. Expression of S1P3 was verified in all cells before capturing images of GFP localization.
Ca2+ Imaging
Changes in intracellular [Ca2+] were determined using the ratiometric calcium indicator dye Fura-2. RH7777 cells were transfected with an S1P3-EGFP fusion expression construct, cultured on collagen-coated coverslips, loaded with Fura-2 acetoxymethyl ester (Fura-2/AM; 8µM) in the presence of 1.5 µM pluronic acid F-127, and incubated for 30–45 min at room temperature in the dark in DMEM. Coverslips were then washed with PBS and secured to the bottom of a laminar flow perfusion chamber RC-25 (Warner Instrument Corporation) with vacuum grease. Cells were selected for absence or presence of S1P3 expression based on EGFP fluorescence. S1P was prepared as described above and applied with a micropipette. Images of Fura-2-loaded cells with the excitation wavelength alternating between 340 nm and 380 nm were captured with a cooled CCD camera every ∼4 sec. The ratio of fluorescence intensity emitted after excitation at 340 nm and 380 nm (F340/F380) was calculated (MetaFluor, Molecular Devices). Cells that exhibited oscillatory fluorescence or elevated F340/F380 ratios (>0.7) prior to S1P stimulation were eliminated from analysis. N = 23–34.
Camp Measurements
S1P3-EGFP over-expressing cell lines were generated by transfecting C6 glioma cells with linearized S1P3-EGFP-pcDNA3.1 using Lipofectamine 200 transfection reagent (Invitrogen, Carlsbad, CA, USA). Stable transfectants were selected using 1 mg/ml of geneticin (Invitrogen, Carlsbad, CA, USA) and clonally expanded. Cells were seeded at 50,000 cells/well in a 24-well plate, serum-starved overnight, pre-treated with 7H9 and S1P as indicated, and stimulated with 0.5 µM forskolin, 0.5 µM 3-isobutyl-1-methylxanthine to elevate basal cAMP. After a 20 minute incubation, cAMP content was determined from cell lysates by ELISA (Cayman Biochemicals, Ann Arbor, MI) per manufacturer’s instructions.
Systemic Inflammation Model
Systemic inflammation experiments were performed on 6 week-old, male C57Bl/6J mice obtained directly from The Jackson Laboratory (Bar Harbor, Maine, USA). Working solutions of normal mouse IgG (cat # 31202, Thermo Fisher Scientific, Rockford, IL, USA) and 7H9 were made in PBS at 0.1 mg/ml, and were administered at 10 µl/g body weight, i.p., 3 times with the following schedule: 12 hours prior, 15 minutes prior, and 24 hours following LPS administration. LPS was prepared in a working solution of 0.8 mg/ml in PBS and administered once at 10 µl/g body weight i.p. Mice were monitored continuously for 1 hour, then 3 times/day for 5 days.
Breast Tumor Xenografts
MCF7 human breast tumor cells were cultured until confluence and the formation of domes, then detached with a rubber policeman and suspended in 50%PBS/50% Matrigel (cat #356234, BD Biosciences, San Jose, CA, USA) at ∼25 × 106 cells/ml. 100µl of cell suspension was delivered subcutaneously into 6 week-old, female NU/J mice (stock # 002019, The Jackson Laboratory, Bar Harbor, Maine, USA) under isoflorane anesthesia. Concurrently, a 60-day release estrogen pellet (cat #SE-121, Innovative Research of America, Sarasota, FL, USA) was implanted at a second subcutaneous site. Tumor development was monitored every ∼2 days by caliper measurement. Tumor volume was calculated by length × width2.
Histology
Tumors were harvested, cut into pieces <10 mm in diameter, fixed in 4% paraformaldehyde overnight, processed and embedded in paraffin using standard techniques, sectioned at 10 µm, and stained with hematoxylin and eosin. Images were captured with a 5X objective. Area of necrotic lesions and total tumor area were determined with Image J software.
Supporting Information
Figure S1.
Immunocytochemical analysis of serum from treated mice demonstrates that 7H9 stably accumulates in serum. Mice were treated with normal IgG (A-C) or 7H9 (D-F) at 1 mg/kg, i.p., QOD for 3 weeks, and serum was collected two days after the last dose. HEK293 cells were transfected with S1P3-GFP and incubated with diluted serum (1∶100). Detection with a Cy3-anti-mouse IgG reveals that S1P3-reactive antibodies are present only in 7H9-treated serum at a concentration of 10–100 µg/ml.
https://doi.org/10.1371/journal.pone.0035129.s001
(TIF)
Figure S2.
Treatment with 7H9 may improve tamoxifen efficacy. MCF7 xenograft tumors were established in a small cohort of nude mice as described in Materials and Methods. (A) Mean tumor volume was reduced by 7H9 (1 mg/kg, i.p., QOD) or tamoxifen (5 mg, 60-day release, s.q.) monotherapies, and by combined treatment. (B) Tumor cell proliferation was evaluated by immunolabeling with mitotic marker, phospho-histone H3 (EMD Millipore, Billerica, MA), and counting positive nuclei under 20X magnification. (C) Area of necrotic lesions was calculated as described in Materials and Methods. N = 3 mice/group, *p<0.05.
https://doi.org/10.1371/journal.pone.0035129.s002
(TIF)
Author Contributions
Conceived and designed the experiments: DRH GLH GBB. Performed the experiments: DRH MBC. Analyzed the data: DRH GLH. Wrote the paper: DRH GLH.
References
- 1. Maggon K (2007) Monoclonal antibody "gold rush". Curr Med Chem 14: 1978–1987.
- 2. Oskouian B, Saba JD (2004) Death and taxis: what non-mammalian models tell us about sphingosine-1-phosphate. Semin Cell Dev Biol 15: 529–540.
- 3. Meyer zu Heringdorf D, Liliom K, Schaefer M, Danneberg K, Jaggar JH, et al. (2003) Photolysis of intracellular caged sphingosine-1-phosphate causes Ca2+ mobilization independently of G-protein-coupled receptors. FEBS Lett 554: 443–449.
- 4. Zhang YH, Vasko MR, Nicol GD (2006) Intracellular sphingosine 1-phosphate mediates the increased excitability produced by nerve growth factor in rat sensory neurons. J Physiol 575: 101–113.
- 5. Hait NC, Allegood J, Maceyka M, Strub GM, Harikumar KB, et al. (2009) Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science 325: 1254–1257.
- 6. Spiegel S, Milstien S (2003) Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 4: 397–407.
- 7. Radeff-Huang J, Seasholtz TM, Matteo RG, Brown JH (2004) G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival. J Cell Biochem 92: 949–966.
- 8. Herr DR, Chun J (2007) Effects of LPA and S1P on the nervous system and implications for their involvement in disease. Current Drug Targets 8: 155–167.
- 9. Choi JW, Herr DR, Noguchi K, Yung YC, Lee CW, et al. (2010) LPA receptors: subtypes and biological actions. Annu Rev Pharmacol Toxicol 50: 157–186.
- 10. Nava VE, Hobson JP, Murthy S, Milstien S, Spiegel S (2002) Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells. Exp Cell Res 281: 115–127.
- 11. Sarkar S, Maceyka M, Hait NC, Paugh SW, Sankala H, et al. (2005) Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7 human breast cancer cells. FEBS Lett 579: 5313–5317.
- 12. Doll F, Pfeilschifter J, Huwiler A (2005) The epidermal growth factor stimulates sphingosine kinase-1 expression and activity in the human mammary carcinoma cell line MCF7. Biochim Biophys Acta 1738: 72–81.
- 13. Benaud C, Oberst M, Hobson JP, Spiegel S, Dickson RB, et al. (2002) Sphingosine 1-phosphate, present in serum-derived lipoproteins, activates matriptase. J Biol Chem 277: 10539–10546.
- 14. Skoura A, Sanchez T, Claffey K, Mandala SM, Proia RL, et al. (2007) Essential role of sphingosine 1-phosphate receptor 2 in pathological angiogenesis of the mouse retina. J Clin Invest 117: 2506–2516.
- 15. Liu Y, Wada R, Yamashita T, Mi Y, Deng CX, et al. (2000) Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J Clin Invest 106: 951–961.
- 16. Visentin B, Vekich JA, Sibbald BJ, Cavalli AL, Moreno KM, et al. (2006) Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple tumor lineages. Cancer Cell 9: 225–238.
- 17. Goetzl EJ, Dolezalova H, Kong Y, Zeng L (1999) Dual mechanisms for lysophospholipid induction of proliferation of human breast carcinoma cells. Cancer Res 59: 4732–4737.
- 18. Dolezalova H, Shankar G, Huang MC, Bikle DD, Goetzl EJ (2003) Biochemical regulation of breast cancer cell expression of S1P2 (Edg-5) and S1P3 (Edg-3) G protein-coupled receptors for sphingosine 1-phosphate. J Cell Biochem 88: 732–743.
- 19. Yamashita H, Kitayama J, Shida D, Yamaguchi H, Mori K, et al. (2006) Sphingosine 1-phosphate receptor expression profile in human gastric cancer cells: differential regulation on the migration and proliferation. J Surg Res 130: 80–87.
- 20. Arikawa K, Takuwa N, Yamaguchi H, Sugimoto N, Kitayama J, et al. (2003) Ligand-dependent inhibition of B16 melanoma cell migration and invasion via endogenous S1P2 G protein-coupled receptor. Requirement of inhibition of cellular RAC activity. J Biol Chem 278: 32841–32851.
- 21.
Sugimoto N, Takuwa N, Okamoto H, Sakurada S, Takuwa Y (2003) Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform. Mol Cell Biol 23: 1534–1545.
- 22. Le Stunff H, Mikami A, Giussani P, Hobson JP, Jolly PS, et al. (2004) Role of sphingosine-1-phosphate phosphatase 1 in epidermal growth factor-induced chemotaxis. J Biol Chem 279: 34290–34297.
- 23.
Martin JL, Lin MZ, McGowan EM, Baxter RC (2009) Potentiation of growth factor signaling by insulin-like growth factor binding protein-3 in breast epithelial cells requires sphingosine kinase activity. Journal of Biological Chemistry.
- 24. Sukocheva O, Wadham C, Holmes A, Albanese N, Verrier E, et al. (2006) Estrogen transactivates EGFR via the sphingosine 1-phosphate receptor Edg-3: the role of sphingosine kinase-1. J Cell Biol 173: 301–310.
- 25. Lee MJ, Thangada S, Claffey KP, Ancellin N, Liu CH, et al. (1999) Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 99: 301–312.
- 26. Wang F, Van Brocklyn JR, Hobson JP, Movafagh S, Zukowska-Grojec Z, et al. (1999) Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis. J Biol Chem 274: 35343–35350.
- 27.
Kimura T, Watanabe T, Sato K, Kon J, Tomura H, et al. (2000) Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3. Biochem J 348 Pt 1: 71–76.
- 28. Paik JH, Chae S, Lee MJ, Thangada S, Hla T (2001) Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3- and beta1-containing integrins. J Biol Chem 276: 11830–11837.
- 29. Langlois S, Gingras D, Beliveau R (2004) Membrane type 1-matrix metalloproteinase (MT1-MMP) cooperates with sphingosine 1-phosphate to induce endothelial cell migration and morphogenic differentiation. Blood 103: 3020–3028.
- 30. Kono M, Mi Y, Liu Y, Sasaki T, Allende ML, et al. (2004) The sphingosine-1-phosphate receptors S1P1, S1P2, and S1P3 function coordinately during embryonic angiogenesis. J Biol Chem 279: 29367–29373.
- 31. Fieber CB, Eldridge J, Taha TA, Obeid LM, Muise-Helmericks RC (2006) Modulation of total Akt kinase by increased expression of a single isoform: requirement of the sphingosine-1-phosphate receptor, Edg3/S1P3, for the VEGF-dependent expression of Akt3 in primary endothelial cells. Exp Cell Res 312: 1164–1173.
- 32. Watson C, Long JS, Orange C, Tannahill CL, Mallon E, et al. (2010) High expression of sphingosine 1-phosphate receptors, S1P1 and S1P3, sphingosine kinase 1, and extracellular signal-regulated kinase-1/2 is associated with development of tamoxifen resistance in estrogen receptor-positive breast cancer patients. American Journal of Pathology 177: 2205–2215.
- 33. Niessen F, Schaffner F, Furlan-Freguia C, Pawlinski R, Bhattacharjee G, et al. (2008) Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation. Nature 452: 654–658.
- 34. Nixon GF (2009) Sphingolipids in inflammation: pathological implications and potential therapeutic targets. British Journal of Pharmacology 158: 982–993.
- 35. Oz-Arslan D, Ruscher W, Myrtek D, Ziemer M, Jin Y, et al. (2006) IL-6 and IL-8 release is mediated via multiple signaling pathways after stimulating dendritic cells with lysophospholipids. J Leukoc Biol 80: 287–297.
- 36. Czeloth N, Bernhardt G, Hofmann F, Genth H, Forster R (2005) Sphingosine-1-phosphate mediates migration of mature dendritic cells. Journal of Immunology 175: 2960–2967.
- 37. Peters SL, Alewijnse AE (2007) Sphingosine-1-phosphate signaling in the cardiovascular system. Curr Opin Pharmacol 7: 186–192.
- 38.
Roviezzo F, Brancaleone V, De Gruttola L, Vellecco V, Bucci M, et al. (2011) Sphingosine-1-phosphate modulates vascular permeability and cell recruitment in acute inflammation in vivo. Journal of Pharmacology and Experimental Therapeutics.
- 39.
Singleton PA, Moreno-Vinasco L, Sammani S, Wanderling SL, Moss J, et al. (2007) Attenuation of vascular permeability by methylnaltrexone: role of mOP-R and S1P3 transactivation. American Journal of Respiratory Cell and Molecular Biology 37: 222–231.
- 40. Singleton PA, Dudek SM, Ma SF, Garcia JG (2006) Transactivation of sphingosine 1-phosphate receptors is essential for vascular barrier regulation. Novel role for hyaluronan and CD44 receptor family. Journal of Biological Chemistry 281: 34381–34393.
- 41. Ruf W, Furlan-Freguia C, Niessen F (2009) Vascular and dendritic cell coagulation signaling in sepsis progression. J Thromb Haemost 7: Suppl 1118–121.
- 42. Ferguson SS, Caron MG (2004) Green fluorescent protein-tagged beta-arrestin translocation as a measure of G protein-coupled receptor activation. Methods Mol Biol 237: 121–126.
- 43. Licht T, Tsirulnikov L, Reuveni H, Yarnitzky T, Ben-Sasson SA (2003) Induction of pro-angiogenic signaling by a synthetic peptide derived from the second intracellular loop of S1P3 (EDG3). Blood 102: 2099–2107.
- 44. Kon J, Sato K, Watanabe T, Tomura H, Kuwabara A, et al. (1999) Comparison of intrinsic activities of the putative sphingosine 1-phosphate receptor subtypes to regulate several signaling pathways in their cDNA-transfected Chinese hamster ovary cells. Journal of Biological Chemistry 274: 23940–23947.
- 45. Ishii I, Ye X, Friedman B, Kawamura S, Contos JJ, et al. (2002) Marked perinatal lethality and cellular signaling deficits in mice null for the two sphingosine 1-phosphate (S1P) receptors, S1P(2)/LP(B2)/EDG-5 and S1P(3)/LP(B3)/EDG-3. J Biol Chem 277: 25152–25159. Epub 22002 May 25152:
- 46. Ishii I, Friedman B, Ye X, Kawamura S, McGiffert C, et al. (2001) Selective loss of sphingosine 1-phosphate signaling with no obvious phenotypic abnormality in mice lacking its G protein-coupled receptor, LP(B3)/EDG-3. J Biol Chem 276: 33697–33704. Epub 32001 Jul 33696:
- 47. Jacobs E, Watson SA, Michaeli D, Ellis IO, Robertson JF (1999) Anti-gonadotrophin releasing hormone antibodies inhibit the growth of MCF7 human breast cancer xenografts. British Journal of Cancer 80: 352–359.
- 48. Kato Y, Okollie B, Raman V, Vesuna F, Zhao M, et al. (2007) Contributing factors of temozolomide resistance in MCF-7 tumor xenograft models. Cancer Biol Ther 6: 891–897.
- 49. Li Z, Carrier L, Belame A, Thiyagarajah A, Salvo VA, et al. (2009) Combination of methylselenocysteine with tamoxifen inhibits MCF-7 breast cancer xenografts in nude mice through elevated apoptosis and reduced angiogenesis. Breast Cancer Res Treat 118: 33–43.
- 50. Chun J, Brinkmann V (2011) A mechanistically novel, first oral therapy for multiple sclerosis: the development of fingolimod (FTY720, Gilenya). Discov Med 12: 213–228.
- 51. Sukocheva O, Wadham C, Holmes A, Albanese N, Verrier E, et al. (2006) Estrogen transactivates EGFR via the sphingosine 1-phosphate receptor Edg-3: the role of sphingosine kinase-1. Journal of Cell Biology 173: 301–310.
- 52. Budde K, Schmouder RL, Brunkhorst R, Nashan B, Lucker PW, et al. (2002) First human trial of FTY720, a novel immunomodulator, in stable renal transplant patients. J Am Soc Nephrol 13: 1073–1083.
- 53. Kappos L, Antel J, Comi G, Montalban X, O'Connor P, et al. (2006) Oral fingolimod (FTY720) for relapsing multiple sclerosis. N Engl J Med 355: 1124–1140.
- 54. LaMontagne K, Littlewood-Evans A, Schnell C, O'Reilly T, Wyder L, et al. (2006) Antagonism of sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization. Cancer Res 66: 221–231.
- 55. Pchejetski D, Bohler T, Brizuela L, Sauer L, Doumerc N, et al. (2010) FTY720 (fingolimod) sensitizes prostate cancer cells to radiotherapy by inhibition of sphingosine kinase-1. Cancer Research 70: 8651–8661.