CD36-mediated activation of endothelial cell apoptosis by an N-terminal recombinant fragment of thrombospondin-2 inhibits breast cancer growth and metastasis in vivo
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Thus far the clinical benefits seen in breast cancer patients treated with drugs targeting the vascular endothelial growth factor (VEGF) pathway are only modest. Consequently, additional antiangiogenic approaches for treatment of breast cancer need to be investigated. Thrombospondin-2 (TSP-2) has been shown to inhibit tumor growth and angiogenesis with a greater potency than the related molecule TSP-1. The systemic effects of TSP-2 on tumor metastasis and the underlying molecular mechanisms of the antiangiogenic activity of TSP-2 have remained poorly understood. We generated a recombinant fusion protein consisting of the N-terminal region of TSP-2 and the IgG-Fc1 fragment (N-TSP2-Fc) and could demonstrate that the antiangiogenic activity of N-TSP2-Fc is dependent on the CD36 receptor. We found that N-TSP2-Fc inhibited VEGF-induced tube formation of human dermal microvascular endothelial cells (HDMEC) on matrigel in vitro and that concurrent incubation of anti-CD36 antibody with N-TSP2-Fc resulted in tube formation that was comparable to untreated control. N-TSP2-Fc potently induced apoptosis of HDMEC in vitro in a CD36-dependent manner. Moreover, we could demonstrate a CD36 receptor-mediated loss of mitochondrial membrane potential and activation of caspase-3 in HDMEC in vitro. Daily intraperitoneal injections of N-TSP2-Fc resulted in a significant inhibition of the growth of human MDA-MB-435 and MDA-MB-231 tumor cells grown in the mammary gland of immunodeficient nude mice and in reduced tumor vascularization. Finally, increased serum concentrations of N-TSP2-Fc significantly inhibited regional metastasis to lymph nodes and distant metastasis to lung as shown by quantitative real-time alu PCR. These results identify N-TSP2-Fc as a potent systemic inhibitor of tumor metastasis and provide strong evidence for an important role of the CD36 receptor in mediating the antiangiogenic activity of TSP-2.
KeywordsBreast cancer Thrombospondin-2 CD36 Metastasis Angiogenesis
There is large amount of evidence that tumor growth is dependent on angiogenesis [8, 15, 16]. Clinically, proof of concept has begun with the recent regulatory approvals of three antiangiogenic therapies targeting the vascular endothelial growth factor (VEGF) pathway. Despite early benefits seen in breast cancer patients treated with anti-VEGF therapies , enduring efficacy with regard to survival benefits is relatively modest . In addition, recent data provided strong evidence that tumors may escape or become resistant to the antiangiogenic effects of blocking the VEGF signaling pathway [14, 36]. Therefore, additional antiangiogenic approaches need to be evaluated. Two members of the thrombospondin (TSP) family [1, 22], TSP-1 and TSP-2, are important naturally occurring angiogenesis inhibitors. Both proteins were shown to have potent antiangiogenic activity by inhibiting endothelial cell proliferation, migration, and tube formation in response to multiple angiogenic stimuli [27, 50], and by inhibiting angiogenic responses in a number of in vivo models . The antiangiogenic activity of TSP-1 was localized to the properdin-like type I repeats, also called thrombospondin structural homology repeats (TSR), in the N-terminal domain of TSP-1 . In contrast to TSP-1, the molecular mechanism of the antiangiogenic activity of TSP-2 has not been as well studied. Since it has been reported that TSP-2 is more effective in inhibiting tumor growth and angiogenesis than TSP-1   , we generated a recombinant fusion protein consisting of the N-terminal region of TSP-2 and an IgG-Fc1 fragment (N-TSP2-Fc). N-TSP2-Fc contained the procollagen homology domain as well as three type I repeats of TSP-2. Here, we report that recombinant N-TSP2-Fc induced apoptosis of endothelial cells by CD36 receptor-mediated loss of mitochondrial membrane potential and activation of caspase-3. In contrast to the majority of experimental antiangiogenic studies that only focused on the effects on primary tumor growth, with much less attention on metastasis, we provide strong evidence that N-TSP2-Fc significantly inhibited tumor growth of xenotransplanted MDA-MB-435 and MDA-MB-231 breast cancer cells and both lymph node as well as lung metastasis of MDA-MB-435 tumor cells grown as xenotransplants in the mammary gland of nude mice.
Materials and methods
Cell lines and culture
Human MDA-MB-435 and MDA-MB-231 breast cancer cells were obtained from ATCC and maintained in DMEM medium (Invitrogen) containing 10% FCS, 4.5 mg/ml glucose, 2 mM l-glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycin. Human 293-EBNA (Epstein-Barr virus nuclear antigen) cells (Invitrogen) were maintained in DMEM/F12 medium (Invitrogen) containing 10% FCS, 15 mM HEPES, 1.0 μg/ml puromycin, 100 units/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml fungizone, amphotericin B (Invitrogen). Human dermal microvascular endothelial cells (HDMEC) from Promocell (Heidelberg, Germany) were cultured in ECGM-MV medium (Promocell) containing fetal calf serum (0.05 ml/ml), endothelial cell growth supplement (0.004 ml/ml), recombinant human epidermal growth factor (10 ng/ml), heparin (22.5 μg/ml), hydrocortisone (1 μg/ml), and phenol red (0.62 ng/ml) supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin.
The coding sequences of the N-terminal region of TSP-2 (aa 18-550 of human TSP-2) and the Fc region of human IgG1 (hinge and CH′′ and CH3 domains) were obtained by PCR and cloned into the the NheI and BamHI sites of a modified pCEP4 expression vector  that contains a cytomegalovirus enhancer/promoter, the EBNA-1 gene, and a puromycin selection cassette. Human 293-EBNA cells were stably transfected with the expression vector using the Fugene transfection reagent (Roche).
Purification of recombinant N-TSP2-Fc
The secreted fusion protein was purified from serum-free culture medium by using affinity chromatography with Protein G Sepharose (GE Healthcare Europe). The fusion protein was eluted with 0.1 M glycine (pH 2.7), and each fraction was neutralized with 1 M Tris–HCl (pH 9.0). After dialysis the protein was stored at −80°C. The protein concentration was determined using the BCA protein assay kit (Pierce).
Tube formation assay
Twenty-four-well plates were coated with 300 μl Matrigel (BD Pharmingen) and incubated for 1 h at 37°C. HDMEC (1 × 105 cells) in ECGM-MV containing VEGF (20 ng/ml) were seeded into duplicate and observed at 12 h. The formation of tube-like structures was analyzed using an Olympus IX51 microscope. Images were captured and computer-assisted analyses of tube length were performed using the Photoshop CS3 software (Adobe Systems).
Primary HDMEC incubated in ECGM containing VEGF165 (R&D Systems) at a concentration of 20 ng/ml were incubated with 40 μg/ml of N-TSP2-Fc with or without concurrent addition of 10 μg/ml of blocking CD36 antibody (Abcam) for 72 h. Apoptosis was evaluated by using a procedure similar to that described by Nicoletti et al. . Briefly, a pellet containing 1 × 106 HDMEC was gently resuspended in 500 ml of hypotonic fluorochrome solution containing 0.1% Triton X-100 (Sigma), 0.1% sodium citrate, and 50 mg/ml propidium iodide (Sigma). After overnight incubation the cell suspensions were assayed by flow cytometry analysis of cellular DNA content on a FACSCalibur equipment (Becton-Dickinson Immunocytometry Systems) using CellQuest software (Becton-Dickinson Immunocytometry Systems).
Mitochondrial membrane potential measurement
Human dermal microvascular endothelial cells cultured in ECGM containing VEGF165 (R&D Systems) at a concentration of 20 ng/ml were incubated with 40 μg/ml of N-TSP2-Fc with or without concurrent addition of 10 μg/ml of blocking CD36 antibody (Abcam) for 72 h. The mitochondrial membrane potential was analyzed using the JC-1 mitochondrial membrane potential detection kit (Biotium).
Western blot analysis of caspase-3 activity
Human dermal microvascular endothelial cells were treated with N-TSP2-Fc (final concentration 40 μg/ml) with or without concurrent addition of 10 μg/ml of blocking CD36 antibody (Abcam) for 72 h. Cell lysates from HDMEC were separated on SDS/PAGE under reducing conditions and transferred to nitrocellulose membranes (HybonD-ECL, GE Healthcare Europe, Munich, Germany) and then immunoblotted with a rabbit anti-human active caspase-3 polyclonal antibody (BD Pharmingen). The specifically bound antibody was detected using the enhanced chemiluminescence kit (Millipore). The bands were analyzed using the Kodak image system (Kodak).
Tumorigenesis and metastasis assay
2 × 106 MDA-MB-435 or 5 × 106 MDA-MB-231 breast cancer cells were injected bilaterally into the second mammary fat pads of athymic, female, 6-week-old NCR nu/nu mice. The tumor volumes were calculated using the following formula: Volume = 4/3 × π × (1/2 × smaller diameter)2 × 1/2 × larger diameter. After euthanizing the mice, tumors, axillary lymph nodes, and lungs were removed from each mouse. The tissue was embedded in OCT compound or was snap frozen in liquid nitrogen. All animal studies were done according to the German ethical guidelines and the German laws for protection of animals.
Mice were intraperitoneally injected with N-TSP2-Fc (100 μg/100 μl PBS). Serum samples were obtained at various times (0, 0.5, 1, 2, 6, 12, and 24 h) after injection. The concentration of TSP-2 was determined using an inhibition ELISA. 96-well plates (MaxiSorbTM, Nunc) were coated overnight with N-TSP2-Fc at a concentration of 1 μg/ml. The remaining reactive sites were blocked with 1% BSA. A polyclonal goat antibody directed against the N-terminal region of human TSP-2 (R&D) was previously mixed in 96-well plates either with standard solutions of N-TSP2-Fc (ranging from 2 pg/ml to 20 μg/ml) or with mouse serum to be tested that was diluted 1:50 and 1:500 with PBS. This mixture was added to the N-TSP2-Fc-coated wells and incubated for 2 h at RT. The samples were assayed using a chemiluminescent substrate (SuperSignal ELISA Pico substrate). Luminescence was measured at 425 nm using a multi-detection microplate reader (BioTek Instruments GmbH). Data were analyzed using the SigmaPlot software (Systat Software), and the concentration of TSP-2 in each sample was calculated from the standard curve.
Analysis of tumor angiogenesis
Cryostat sections (5 μm) were stained with a rat anti-mouse CD31 monoclonal antibody (BD Pharmingen). Representative sections obtained from five tumors were analyzed using an Olympus IX51 microscope. Images were captured and morphometric analyses were performed using the IP LAB software (Scanalytics) as described . Three different fields in each section were examined at 10× magnification, and the number of vessels per mm2, the vessel size, and the relative area occupied by tumor blood vessels within the tumors were determined.
Quantitative analysis of metastasis
The DNEasy Blood and Tissue Kit (Qiagen) was used for isolation of genomic DNA. Primers (Eurogentec) specific for the human alu sequences (sense: 5′-CATGGTGAAACCCCGTCTCTA-3′; and antisense: 5′-GCCTCAGCCTCCCGAGTAG-3′) were used to amplify human alu repeats present in genomic DNA that was extracted from mouse tissue. The internal TaqMan probe for alu (5′-[YY]-ATTAGCCGGGCGTGGTGGC-[BHQ-1]-3′) was 5′-labeled with the reporter fluorescent dye Yakima Yellow (YY) and carried the non-fluorescent quencher dye Black Hole Quencher-1 (BHQ-1). The 18S rRNA control kit (Eurogentec) was used as an internal reference and was labeled with the reporter dye 6-carboxy-fluorescein (FAM) at the 5′ end and the quencher dye 6-carboxy-tetramethyl-rhodamin (TAMRA) at the 3′ end. PCRs were carried out in 25 μl using 2×TaqMan Universal PCR Master Mix (Applied Biosystems), 0.9 μM each alu specific oligonucleotide primer, 250nM internal alu probe, 1.5 μl 18S rRNA primer mix, 0.5 μl internal 18S rRNA probe, and 1 μl genomic DNA. The final concentration for genomic DNA was 5 ng/μl for lymph node tissue and 50 ng/μl for lung tissue. PCR was carried out for 2 min at 50°C and for 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C using the ABI PRISM™ 7700 Sequence Detector (Applied Biosystems). Serial dilution steps (1:10) of genomic DNA obtained from human MDA-MB-435 cells in genomic DNA were obtained from either mouse lymph node or lung tissue served to construct the calibration curves. The cycle at which the alu PCR product becomes detectable above background signal (Ct alu ) was determined for each dilution series. The alu signal was normalized against the relative quantity of 18S rRNA and expressed as ∆Ct = Ct alu − Ct18SrRNA. A standard curve was generated for each measurement by using the real-time PCR assay and by plotting ∆Ct as a function of the relative amount of human DNA in murine DNA (%). The R 2 value (correlation coefficient) was >0.96 for all measurements demonstrating an excellent relationship between the alu signal and the relative amount of human DNA with a detection limit of 0.001% human DNA in murine DNA.
The two-sided unpaired t-test was used to analyze the tumor and metastasis data and to analyze the differences in microvessel density, vessel size, and total vascular area. Tube formation, apoptosis, mitochondrial membrane potential, and caspase-3 activity were analyzed for significant differences by one-way ANOVA followed by Student–Newman–Keuls’ test for comparison of individual groups, after a Bartlett test had shown that variances were homogenous.
N-TSP2-Fc inhibits tube formation of CD36 positive endothelial cells
N-TSP2-Fc induces activation of apoptosis in microvascular endothelial cells
N-TSP2-Fc causes activation of caspase-3 and of the mitochondrial-dependent intrinsic pathway of apoptosis
Treatment of HDMEC with N-TSP2-Fc for 72 h resulted in a significant loss of mitochondrial membrane potential and decreased the mitochondrial membrane potential to 66% of control (=100%; P < 0.001). When HDMEC were treated with CD36 blocking antibody, addition of N-TSP2-Fc failed to induce loss of mitochondrial membrane potential (Fig. 2b). In addition, treatment of HDMEC with N-TSP2-Fc for 72 h significantly induced activation of caspase-3 by more than 100% as compared to PBS control (Fig. 2c, d). The activation of caspase-3 by N-TSP2-Fc was almost completely abrogated by parallel incubation of HDMEC with blocking CD36 antibody (Fig. 2c, d).
Systemic treatment with N-TSP2-Fc inhibits tumor growth and angiogenesis
Intraperitoneal injection of N-TSP2-Fc results in increased serum level of TSP-2
Time (h) after injection (i.p.)
Serum concentration of TSP-2
0.0284 ± 0.0036 μg/ml
16.8 ± 1.8 μg/ml
19.7 ± 3.8 μg/ml
0.198 ± 0.053 μg/ml
0.179 ± 0.043 μg/ml
0.029 ± 0.0063 μg/ml
Systemic treatment with N-TSP2-Fc decreases metastasis
TSP-2 is a potent endogenous inhibitor of tumor growth and angiogenesis [17, 45]. The antitumoral effect of TSP-2 is stronger than the effect of TSP-1 . However, the exact molecular mechanism of the antiangiogenic activity of TSP-2 has not been as well studied as that of TSP-1. TSP-2 shows only low sequence homology to TSP-1 in the procollagen region. Moreover, a recombinant fragment of human TSP-2 that only included the N-terminal globular domain and the procollagen homology domain failed to significantly inhibit tumor growth and angiogenesis . Therefore, the antiangiogenic activity of TSP-2 might map to the properdin-like type 1 repeats , which have been implicated in the antiangiogenic activity of TSP-1 [12, 20, 49]. The antiangiogenic effects within the properdin-like type 1 repeats of TSP-1 are mediated partly through interaction of the CSVTCG sequence with the CD36 receptor on endothelial cells [11, 21, 46].
We produced a recombinant fusion protein, N-TSP2-Fc, that encompasses the N-terminal globular region with all three type 1 repeats containing two CSVTCG sequence motifs within the first and second type 1 repeats. N-TSP2-Fc exerted potent antiangiogenic activity in vitro. We found that N-TSP2-Fc significantly inhibited HDMEC tube formation and induced HDMEC apoptosis. N-TSP2-Fc also resulted in a significant increase of caspase-3 activity and a significant loss of mitochondrial membrane potential in cultured HDMEC. Moreover, blockade of the CD36 receptor almost completely abrogated the reported in vitro effects of N-TSP2-Fc, strongly suggesting that the antiangiogenic activity of N-TSP2-Fc is mediated by CD36-dependent induction of endothelial cell apoptosis and at least in part through activation of the intrinsic apoptotic pathway. This is in agreement with the previously reported binding of TSP-2 to CD36 and lack of inhibition of angiogenesis by TSP-2 in CD36 null mice . The exact molecular mechanisms through which TSP-2 and TSRs induce activation of caspase-3 and promote CD36-mediated apoptosis remain to be elucidated.
We investigated the efficacy of systemic therapy with N-TSP2-Fc on both tumor growth and metastasis. To this end we made use of MDA-MB-435 tumor cells representing a well-established orthotopic model system for the study of breast cancer metastasis. MDA-MB-435 cells form primary tumors when injected into the mammary fat pad of nude mice, and spontaneously metastasize from these tumors to the lymph nodes and lungs [37, 44, 52]. However, in a microarray expression study MDA-MB-435 cells have been reported to cluster with several melanoma cell lines , and it has been speculated that that MDA-MB-435 cell is instead the M14 melanoma cell line, due to an early cross-contamination of cell cultures [9, 38]. MDA-MB-435 cells have been shown to express melanocytic proteins  as well as epithelial markers such as cytokeratin 19 and secrete milk proteins and lipids  suggesting that MDA-MB-435 breast cancer cells may have undergone a lineage infidelity during tumor progression . Indeed, lineage infidelity has been seen in breast carcinomas that express both epithelial and melanoma-associated proteins [33, 35]. Very recently, Chambers  reported that although currently available stocks of the MDA-MB-435 and M14 cell lines were shown to be virtually identical at the karyotype level , the conclusion that M14 and MDA-MB-435 cells are of M14 melanoma origin is inconsistent with the fact that the M14 cell line derived from a male patient with metastatic melanoma  and the female karyotypes of both cell lines at present [9, 38]. Chambers  suggested that the evidence from the literature rather is consistent with cell lines being of MDA-MB-435 breast cancer origin and that the MDA-MB-435 cell line represents a poorly differentiated aggressive breast tumor line with expression of both epithelial and melanocytic markers. In addition, in a recent study on distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines Hollestelle et al.  revealed that the MDA-MB-435 cell line was basal-type. They identified subtype-specific gene mutation profiles revealing that basal-type breast cancers may have a genetic basis similar to melanomas [18, 19]. Hollestelle et al. concluded conclusively that MDA-MB-435 is a breast cancer cell line .
We provide strong evidence that intraperitoneally injected N-TSP2-Fc sufficiently reached the blood circulation as shown by an inhibition ELISA and thereby systemically inhibited MDA-MB-435 tumor progression due to an inhibition of angiogenesis. We could also demonstrate that IgG-Fc did not have any major effects on MDA-MB-435 tumor growth in vivo. In addition, the inhibitory effect of recombinant N-TSP2-Fc on the growth of breast carcinomas could be confirmed by using of another, MDA-MB-231 , breast cancer cell line. Systemic treatment with N-TSP2-Fc also resulted in a significant inhibition of xenotransplanted MDA-MB-231 breast cancer cells. Our findings are consistent with recently reported growth inhibition of xenotransplanted A431 squamous cell carcinomas by intraperitoneal application of a recombinant TSP-2 protein that include TSR sequences . We have combined the accuracy and sensitivity of real-time PCR  with the human specificity of alu sequences [40, 48, 54] for direct quantitative evaluation of metastatic MDA-MB-435 breast cancer cells in lymph nodes and lungs. As early as 2 weeks after tumor cell injection we could detect human tumor cell DNA in lymph node and lung tissue of MDA-M-435 breast tumor-bearing mice. The amount of human DNA in the lungs was much lower than that in the lymph nodes, indicating that lymph node metastasis is an earlier event than distant lung metastasis. This is consistent with the clinical course of most breast cancer diseases [4, 31]. We also found significantly less human tumor cell DNA in the lymph nodes and lungs of N-TSP2-Fc-treated mice as compared to control-treated mice. The inhibitory effect on metastasis was maintained over the entire period of in vivo tumor growth. Even at 5 weeks after tumor cell inoculation, both lymph node and lung metastasis were significantly reduced in N-TSP2-Fc-treated mice.
Controversial findings have been reported regarding the effects of TSP-1 on metastasis [2, 3]  . Recent in vitro [2, 5] and in vivo  data suggest that prometastatic effects of endogenous TSP-1 may involve the activation of transforming growth factor-β (TGF-β) via the unique sequence KRFK found between the first and second type 1 repeats of TSP-1 . Noteworthy, TSP-2 lacks the KRFK sequence, and recent studies have not found any evidence for activation of latent TGF-β by TSP-2 . Whether the lack of TGF-β activation may explain the lack of any reported prometastatic effect of TSP-2 remains to be further evaluated. In support of this hypothesis are recent findings demonstrating that lymph node metastasis was only observed in TSP-2-null mice but not in wild-type mice in a multistep chemically induced skin carcinogenesis protocol  whereas in a transgenic mouse model of breast cancer pulmonary metastasis was increased in TSP-1 null mice as compared to wild-type mice . Recently, preclinical studies reported that angiogenesis inhibitors targeting the VEGF signaling pathway reduced primary tumor growth but promoted tumor invasiveness and metastasis in vivo [14, 36]. Multi-targeting of parallel angiogenesis regulating pathways have been proposed to improve the benefits of antiangiogenic drug therapy . Our results demonstrate that N-TSP2-Fc treatment potently inhibits both tumor growth and metastasis in vivo mediated by CD36-dependent inhibition of tumor angiogenesis, and they indicate its potential use for antiangiogenic combination therapies to treat breast cancer.
We thank Renate Dietrich, Matthias Läsche, and Hiltrud Schulz for excellent technical assistance. This study was supported by Deutsche Forschungsgemeinschaft grants (HA 2898 4-1 and 4-2, MK SFB 832 A8).
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