Introduction

Cyr61 (CCN1), as the first cloned member of cysteine-rich protein (CCN) family, is a secreted, cysteine-rich, heparin-binding extracellular matrix-associated protein [1, 2]. To date, a number of reports describe that Cyr61 is involved in many cell biological functions. For example, Cyr61 has been identified to mediate cell adhesion, migration, proliferation, apoptosis, and angiogenesis [3–6].

As its widely spreading integrin receptors, Cyr61 located in different tissues and may play different roles [5, 7, 8]. Previous studies in tumors pathogenesis revealed that overexpression of Cyr61 is related to the development of neovascularization and tumor formation [9–13]. Among these studies, the expression profile of Cyr61 in breast cancer is reported intensively [5, 14–17]. For example, overexpression of Cyr61 was found in 70% of breast cancer patients with infiltrating ductal carcinomas [7]. In some reports, Cyr61 was localized exclusively for hyperplastic ductal epithelial cells [5, 7, 14]. Moreover, some studies showed that overexpression of Cyr61 was related to lymph node metastasis [15, 17–20]. According to these findings, it is hypothesized that overexpressing Cyr61 might involved in breast cancer growth and metastasis. Consistently, Cyr61 high-expression breast cancer cell line MDA-MB-231 showed faster growth and more severe metastasis than Cyr61 low-expression cell line MCF-7 in vitro and in vivo, when Cyr61 overexpressed in MCF-7 increased the cells invasion, migration, and proliferation ability [7, 16, 21, 22]. These indicated that blockage of Cyr16 might be a potent target for breast cancer treatment.

Like other members of CCN family, Cyr61 is composed of an N-terminal secretary peptide followed by four structural domains that include (1) an insulin-like growth factor–binding protein homology domain (IGFBP domain), (2) a von Willebrand factor type C domain (VWC domain), (3) a thrombospondin type 1 repeat homology domain (TSP domain), and (4) a C-terminal domain (CT domain). It possesses heparin-binding motifs and sequence similarity to the C-termini of von Willebrand factor and mucin [3]. The structure and function of CCN family have been studied intensively [1, 23–26]. In breast cancer research, reports implicated that the domain 4 of Cyr61 was important for promoting tumor cell proliferation, migration, and invasion [27]. These results indicated that prominent expression of Cyr61 contributed to breast cancer development, where blocking or downregulation of Cyr61 might be benefit for breast cancer therapy [20–22].

Given its role in breast cancer cells’ growth and metastasis, Cyr61 might be a candidate target for human breast cancer treatment. In this study, using anti-Cyr61 monoclonal antibody generated in our laboratory, we examined Cyr61 expression profile among Chinese breast cancer patients with different clinical stages, and the results showed that Cyr61 was overexpressed in patients as previous reports [7]. Further, we examined the role of Cyr61 in tumorigenesis of MDA-MB-231 cells using siRNA and anti-Cyr61 monoclonal antibody 093G9. Moreover, we showed that 093G9 could inhibit MDA-MB-231 migration and invasion in vitro consistent with upregulation of MMPs inhibitor TIMP1 and TIMP2. In addition, using nude mice tumor model, we showed 093G9 could arrest MDA-MB-231 growth and lymph node metastasis in vivo. Furthermore, peptide LPNLGCP located in VWC domain of Cyr61 was identified as an epitope recognized by 093G9 using Ph.D.-C7C phage library display analysis. This suggested that LPNLGCP of VWC domain in Cyr61 protein might be very important in breast cancer cells proliferation, migration, and invasion. Taken together, we provided a solid evidence of inhibition of growth and lymph node metastasis in breast cancer by blockade of Cyr61, which promised Cyr61 as a target for breast cancer treatment.

Materials and methods

Cell lines and animals

Two human breast cancer cell lines, MCF-7 and MDA-MB-231, were obtained from the American Type Culture Collection (ATCC). Five-week-old female nude mice and BALB/c mice were obtained from the Shanghai Laboratory Animal Center, Chinese Academy of Science. Mice were housed in pathogen-free conditions and were treated in accordance with the guideline of the Committee on Animals of the Shanghai Jiaotong University School of Medicine.

Production of monoclonal antibody

The 093G9 and 096B7 hybridoma cell lines secreting mouse monoclonal antibody (mAb) against human Cyr61 were generated by the standard hybridoma technique in our laboratory [28]. The mouse mAbs, 093G9 (IgG1, κ) and 096B7 (IgG1, κ), were purified by Protein A affinity chromatograph. The purity of mAbs 093G9 and 096B7 was confirmed by HPLC.

Indirect ELISA

The recombinant human Cyr61 (hCyr61) was diluted to a concentration of 1 μg/ml with 0.05 M Tris–Hcl buffer (pH 8.0) and coated on a 96-well plate with 100 μl/well, 4°C overnight. After washing the plates three times with phosphate-buffered saline (PBS), 1% BSA containing 0.05% Tween 20 in PBS was added to the well for blocking for 2 h at 37°C to avoid non-specific binding. After washing the plates three times, the anti-Cyr61 mouse monoclonal antibody was added and incubated for 1 h at 37°C. After washing again, HRP-conjugated goat anti-mouse IgG (R&D) was added and incubated for 1 h at 37°C. The plate was washed again and incubated with 100 μl of tetramethyl benzidine (TMB) substrate at room temperature for 15 min. After stopping the reaction with 2 M H2SO4, the absorbance was measured at 450 nm.

Characteristics of mAbs

Titers of culture supernatant and ascites form of hybridoma cells were detected by indirect ELISA after serial dilutions. The isotypes of the immunoglobulins were analyzed using the Mouse Immunoglobulin Isotype Panel kit (SouthernBiotech, AL, USA), according to the manufacturer’s instructions. The concentrations of the obtained mAbs were measured by Mouse IgG ELISA Quantitation kit (BETHYL, USA), according to the manufacturer’s instructions.

Biacore analysis

The kinetic parameters of anti-Cyr61 antibodies for Cyr61 were determined using the Biacore T100 instrument (Biacore AB, Uppsala, Sweden) according to the manufacturer’s instructions. Briefly, covalent immobilization of purified recombinant human Cyr61 onto a Series S CM5 sensor chip (Biacore) was performed using an amine coupling kit (Biacore). Twofold serial dilutions of anti-Cyr61 monoclonal antibodies (mAbs) were injected over the sensor chip surface at a flow rate of 50 μl/min. Cyr61 irrelevant antibodies were used as controls. The constants of association (Ka) and dissociation (Kd) rate of anti-Cyr61 mAbs were determined with the Biacore T100 Evaluation software version 2.0 (Biacore).

Tissue microarray (TMA) and immunohistochemistry (IHC) assay

The breast cancer TMA was supplied by Xi’an Alena Biotechnology Ltd., Co. (Xi’an, China). IHC studies were performed with a standard EliVision™ method. In brief, TMA sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 min. For antigen retrieval, TMA slides were microwave-treated in 10 mM citrate buffer (pH 6.0) for 10 min. The slides were incubated with mouse anti-Cyr61 mAb (1:200 dilution) overnight at 4°C. The slides were then incubated with a HRP-conjugated goat anti-mouse polyclonal antibody for 60 min and subsequently reacted with DAB. The nucleus was counterstained with hematoxylin. Negative controls were performed by replacing the primary antibody with mouse IgG. To evaluate the IHC staining of Cyr61 in breast cancer TMA, a semi-quantitative scoring criteria for IHC of Cyr61 was used, in which both staining intensity and positive areas were recorded as − (negative), + (weak positive), ++ (moderate positive), and +++ (strong positive), respectively.

RNA extraction and real-time PCR

Real-time PCR was performed as previously reported [8]. The sequence of primers were as follows: GAPDH, forward 5′-GTGAAGGTCGGAGTCAACG-3′ and reverse 5′-TGAGGTCAATGAAGGGGTC-3′; Cyr61, forward 5′-TCCAGCCCAACTGTA AACATCA-3′ and reverse 5′-GGACACAGAGGAATGCAGCC-3′; TIMP1, forward 5′-GGTGGGTGGATGAGTAATGCA-3′ and reverse 5′-AGCGGGTGCGG AAACC-3′; TIMP2, forward 5′-GGGCCAAAGCGGTCAGT-3′ and reverse 5′-TAGGGTTGCCATAAATGTCGTTT-3′.

Western blotting analysis

Both Cyr61 expression in MDA-MB-231, MCF-7 cells and the rhCyr61 protein were detected by Western blotting analysis with special anti-human Cyr61 mAbs 093G9, 096B7 and commercial anti-human Cyr61 mAb (R&D, MN, USA). The activation of AKT and ERK (ERK1/ERK2) was analyzed using specific phosphorylated antibodies (Cell Signaling Technology Inc, Beverly, MA, USA). In briefly, lysates were loaded and run electrophoresis followed by transferring to PVDF membranes (Millipore, MA, USA) at 60v for 2 h. The membranes were blocked with 5% non-fat milk, washed with PBST, and incubated with mAbs at 4°C overnight. Then, the membranes were incubated with HRP-conjugated goat anti-mouse IgG at room temperature for 45 min followed by washing with PBS. The target proteins were examined with ECL system (Millipore, MA, USA) and visualized with autoradiography film.

Immunofluorescence assay

A total of 1 × 105 cells seeded on glass coverslips and grown under standard conditions for 24 h were rinsed in PBS and fixed with 4% paraformaldehyde for 20 min. After permeabilizing with 0.1% Triton X-100 (Sigma, MO, USA) in PBS, cells were blocked with 10% normal goat serum for 1 h. The mouse anti-cyr61 mAb: 096B7 or 093G9 were applied at 1:400 dilutions at 4°C overnight. Alexa Fluor 488-conjugated anti-mouse IgG (ZyMed, CA, USA) was used as a secondary antibody at 1:1,000 dilution for 1 h. Nuclei were counterstained with DAPI (ZyMed, CA, USA) for 3 min. Coverslips with antifade fluorescent mounting medium were transferred onto glass slides and sealed with nail polish.

Cyr61 gene expression knockdown by RNAi

Cyr61 gene knockdown was performed as previously reported [8]. In brief, MDA-MB-231 cells were cultured in 12-well plates. A transfection mixture of siRNA oligonucleotides and Lipofectamine 2000 reagent (Invitrogen, CA, USA) in serum-free medium was added to medium-aspirated cells for 4 h. Then, the medium was replaced with complete DMEM containing 10% FCS for another 24 h incubation.

Cell division and proliferation analysis

MDA-MB-231 cells were labeled with CFSE, and cell division was detected by flow cytometry after Cyr61 gene knocked down. Proliferation was measured by thymidine (3H-TdR) incorporation. In briefly, MDA-MB-231 cells (1.2 × 104 per well) were co-cultured with 093G9, 096B7, or IgG1 isotype control mAb (control IgG) at final concentration of 5, 10, 20 μg/ml, respectively. Cells were pulsed with 1 μCi thymidine (Amersham Biosciences, Piscataway, NJ, USA) for another 16 h. Incorporated radioactivity was measured as c.p.m (Count per min) using a β-plate counter.

Cell migration and invasion assay

Migration assay was performed in a 24-well Transwell tissue culture plate (Costar Corning, MA, USA) with a polycarbonate filter (pore size 5 μm). A total of 5 × 104 cells/well were seeded in upper chambers with 200 μl 0.1% BSA DMEM, and 800 μl supernatant from MDA-MB-231 cells containing secreted Cyr61 as chemoattractant was put at bottom chambers. Then, cells were allowed to migrate in the presence of 093G9 or control IgG at a concentration of 5, 10, and 20 μg/ml for 4 h, respectively, followed by fixed filters with 4% paraformaldehyde for 15 min at room temperature. The cells remained on fixed filters were observed under microscope and counted using 100 ocular grids to quantitative migrated cells. Total cells on six random fields per filter were photographed and counted as migrated cells. For invasion assay, similar for migration assay except (1) transwell insert chambers with 8-μm pore filters were coated with a final concentration of 1 mg/ml of Matrigel (Becton–Dickinson) and (2) cells in upper chambers were allowed to migrate for 24 h. Percent inhibition of migration or invasion = 1 − (cells on fixed filter divided total cells seeded in upper chambers) × 100%.

Animal tumor model and mAbs treatment

A total of 5 × 106 MDA-MB-231 cells were inoculated into 5-week-old female nude mice subcutaneously. Two days later, inoculated mice were randomly assigned to two groups receiving 093G9 and control IgG (5 mg/kg body weight) i.p., twice per week, respectively. Tumor size was measured with caliper once every 3 days until the mice of control group were evaluated as dead after tumors reached a size of 1 cm3 as previous reports. For lymph node metastatic analysis, 2 × 106 MDA-MB-231 cells were injected into the claw pad followed by 093G9 and control IgG treatment same as described earlier. Thirty-five days later, the mice were killed and lymph node metastasis was observed. Tumor volume (mm3) = 0.5 × length × width2. At the end of the experiment, the mice were killed and primary tumors were dissected, weighed, and fixed in 10% formalin for Cyr61 expression analysis with H&E staining and IHC assay.

Biopanning against anti-hCyr61 monoclonal antibody assay

For biopanning epitopes of two anti-Cyr61 monoclonal antibodies, Ph.D.-C7C or Ph.D.-12 phage library displaying assay kits (NEB, MA, USA) were used and panning performed according to the manufacture’s instructions. In brief, microplate wells were coated with mAbs 093G9 or 096B7 (100 μg/well) and blocked with 0.5% BSA (bovine serum albumin) in PBS. The phage library (1.0 × 1011 plaque forming units) was added to the wells, incubated for 1 h, and washed with TBS-0.1% Tween 20. ER2738 Escherichia coli were added and cultured for phage propagation. Phages recovered from the culture supernatant with polyethylene glycol precipitation were used for the next round of panning. After 3 rounds of panning, the phages were cloned on the culture plates for the formation of phage plaques. The positive phage clones were subjected to DNA sequencing and epitope screening.

Statistical analysis

Group measures were shown as mean ± SEM. A Student’s t test was used to analyze the differences between the groups. One-way ANOVA was initially performed to determine whether there was an overall statistically significant change followed by the two-tailed paired or unpaired Student’s t test. P value of <0.05 was considered statistically significant.

Results

Cyr61 high expression in breast cancer tissue

Using TMA and IHC assays, we examined Cyr61 expression in breast cancer tissue obtained from 78 Chinese patients with different clinical stages and pathological types. In breast cancer tissues, Cyr61 was found strong expression in both breast cancer cells and ductal epidermal cells, while in normal breast tissues, Cyr61 was only expressed in ductal epidermal cells (Supplement Figure). Table 1 shows that of the breast cancers studied, 4 were negative or equivocal for Cyr61 expression. Cyr61 protein was expressed in all different clinical stages, including patients with infiltrating breast cancer, lymph node metastasis, and ovarian metastasis (Table 1). This result was consistent with previous reports: Cyr61 indeed overexpressed in breast cancer regardless in primary or in metastatic breast cancer [7, 11, 15, 18].

Table 1 Expression of Cyr61 in breast cancer tissues
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Characterization of anti-Cyr61 mAbs 093G9 and 096B7

In a set of Biacore experiments, the affinity of 093G9 and of 096B7 to human Cyr61 was determined. Twofold dilutions of purified human Cyr61 (1–64 nM) were injected into the flow cell containing immobilized mAbs 093G9 or 096B7. Based on the experimental data, association and dissociation curves were calculated (black lines) and fitted to the recorded curves (colored lines). Association (Ka) and dissociation rate constants (Kd) were determined to be Ka = 1.019 × 106 and Kd = 0.00992 for 096B7, and Ka = 3.125 × 105 and Kd = 0.001952 for 093G9, respectively. The affinity constant Kd was calculated from Kd and Ka to be 9.7 × 10−9 and 6.2 × 10−9 (Fig. 1a). This result indicated that 093G9 and 096B7 showed high antigen-binding activity to Cyr61. The purity of mAbs 096B7 and 093G9 was more than 95% determined by HPLC (Fig. 1b). ELISA showed the high titers of 093G9 and 096B7 binding Cyr61, which were similar to a commercial available one (R&D) (Fig. 1c). The specificity of mAbs was analyzed by Western blot. As shown in Fig. 1d, mAbs 093G9 and 096B7 could bind to human Cyr61 extracted from MDA-MB-231 cells and rhCyr61 specifically as the R&D antibody did.

Fig. 1
figure 1

Characterization of anti-Cyr61 mAbs 093G9 and 096B7. a Cyr61 was injected at twofold increasing concentrations from 1 to 64 nM (curves bottom–top) into the flow cell containing immobilized mAbs 093G9 or 096B7. Calculated curves (black) were superimposed on recorded curves (colored). The affinity constant Kd was calculated from Kd to Ka. b The purity of mAbs 096B7 and 093G9 was determined by HPLC. c ELISA. Serial log dilutions of 096B7, 093G9, or R&D antibodies were added to ELISA plates precoated with human Cyr61 and incubated. After washing, mAbs binding to Cyr61 were detected with HRP-conjugated goat anti-mouse IgG. d 096B7, 093G9, and R&D antibodies bind to rhCyr61 protein and human Cyr61 protein detected by Western blot assays

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Knockdown Cyr61 reduces division of cell line MDA-MB-231

As known that Cyr61 high expressed breast cancer cell line MDA-MB-231 grows faster and increases metastasis compared with low-expressed Cyr61 cell line MCF-7, we first tested Cyr61 expression profiles with our anti-Cyr61 monoclonal antibodies (mAbs) 093G9 and 096B7. The results showed that both mAbs could bind to nature Cyr61 expressed in MDA-MB-231 by Western blot and immunofluorescence staining assay (Fig. 2a, b). Further, to address whether Cyr61 involved in proliferation of MDA-MB-231, Cyr61 expression was inhibited by RNAi technique. The results showed when Cyr61 expression was downregulated up to 60% by siRNA (Fig. 2c), the division of MDA-MB-231 was reduced more than 4 generations (Fig. 2d). These results demonstrated that Cyr61 was very important in MDA-MB-231 cells division.

Fig. 2
figure 2

Cyr61 gene knockdown reduced MDA-MB-231 cells division. a Cyr61 expression in MCF-7 and MDA-MB-231 cells. Upper panel real-time PCR evaluation of Cyr61 expression. Lower panel Western blot analysis of Cyr61 expression. b Immunofluorescence assay shows mAb 093G9 can bind to natural Cyr61 protein in cells. c Cyr61 mRNA was downregulated by special RNAi in MDA-MB-231 cells. d MDA-MB-231 cells division reduced remarkably after RNAi. The peaks in different colors represent the passages of MDA-MB-231 cells labeled with CFSE. SiNC: negative control. SiCyr61: Cyr61 gene was knockdown in MDA-MB-231 cells

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Anti-Cry61 antibody 093G9 inhibits MDA-MB-231 proliferation in vitro and in vivo

To further address whether Cyr61 involved in MDA-MB-231 cell’s growth, we examined proliferation of MDA-MB-231 treated with anti-Cyr61 mAbs 093G9 and 096B7, respectively. The results showed that only 093G9 could inhibit MDA-MB-231 cells proliferation significantly with a dose-dependent manner (Fig. 3a), whereas 096B7 did not show inhibitory capability. To understanding the signaling pathway involved in this activity, downstream protein phosphorylation profiles after Cyr61 mAb neutralization were analyzed. When Cyr61 on MDA-MB-231 was neutralized by 093G9, phosphorylated AKT and ERK decreased remarkably (Fig. 3b). Moreover, to address whether 093G9 could inhibit MDA-MB-231 growth in vivo, we treated nude mice bearing MDA-MB-231 with 093G9. The results showed that tumor volume in the mice received 093G9 was significantly smaller than those treated with control IgG (Fig. 3c, left panel), which consequently 093G9-treated mice had longer life span (Fig. 3c, right panel). Meanwhile, IHC analysis revealed that 093G9-treated tumor cells showed weak Cyr61 expression compared with its counterpart that received control IgG (Fig. 3d). These results implicated that 093G9 could block the biological activity of Cyr61 on cell proliferation in vitro and in vivo by downregulation of AKT and ERK phosphorylation.

Fig. 3
figure 3

mAb 093G9 inhibits MDA-MB-231 cells’ growth in vitro and in vivo. a Decreased proliferation of MDA-MB-231 cells treated with mAbs labeled on axis. Open bars represents 5 μg/ml mAbs were added. Shadow bars mean 10 μg/ml mAbs were used. Black bars represents 20 μg/ml mAbs were added. b Phosphorylated AKT and ERK extracted from MDA-MB-231 cells co-cultured with mAb 093G9 or Con-IgG at different time. c Tumor volume (left panel) and percent survival (right panel) treated with mAb 093G9 and Con-IgG. Open circle represented mice treated with Con-IgG (n = 10). Solid circle represented the mice received 093G9 (n = 10). d H&E staining and immunohistochemical staining of Cyr61 expression in tumor tissue treated with Con-IgG or 093G9 at an original magnification ×200. The data represent mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01

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093G9 inhibits MDA-MB-231 migration and invasion in vitro and in vivo

As many reports presumed that Cyr61 may mediate not only breast cancer cells’ growth but also metastasis and invasion, whether 093G9 could inhibit MDA-MB-231 migration and invasion was analyzed using transwell system. The results indicated that 093G9 inhibited MDA-MB-231 migration and invasion in a dose-dependent manner (Fig. 4a). When treated MDA-MB-231 cells with 20 μg/ml 093G9, the inhibition percent of migration reached to 70% and that of invasion was 60%, respectively (Fig. 4b). Because previous reports implicated that MMPs involved in invasion and metastasis, we detected MMPs inhibitors, TIMP1 and TIMP2, expression in MDA-MB-231 cells treated with 093G9 or control IgG. The result showed that TIMP1 and TIMP2 transcription in 093G9-treated MDA-MB-231 cells was upregulated significantly (Fig. 4c). Furthermore, using nude mice footpad inoculation model, we found that events of lymph node metastasis were significantly less in 093G9-treated group (20%) compared with that received control IgG (60%), consistently, metastatic tumor volume was significantly larger in control group (Fig. 4d). In contrast, 096B7 did not inhibit migration and invasion of MDA-MB-231 cells both in vitro and in vivo (data not shown). These demonstrated that neutralizing Cyr61 by 093G9 could inhibit MDA-MB-231 cells migration and invasion effectively both in vitro and in vivo. Upregulation of TIMP1 and TIMP2 (MMPs inhibitors) might contribute to inhibitory activity of 093G9.

Fig. 4
figure 4

mAb 093G9 inhibits MDA-MB-231 cells migration and invasion in vitro and lymph node metastasis in vivo. a Transwell system assay for MDA-MB-231 cells migration ability (top panel) and invasion ability (bottom panel). Cells on filters were fixed and stained with gentian violet, and then, six random fields per filter were photographed and counted. b Percent inhibition of migration (solid circles) and invasion (open circles) after treated with mAb 093G9 compared with control IgG. c The relative expression of TIMP1 and TIMP2. Open bar represented expression profile treated with Con-IgG, and black bar represented expression profile treated with 093G9. d Lymph node metastasis profile. Tumor volume (left panel) and percent metastasis (right panel) treated with mAb 093G9 or Con-IgG. Open circle or bar represented mice treated with Con-IgG (n = 10). Solid circle or bar represented the mice received 093G9 (n = 10). The data represent mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01

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Mapping of Cyr61 epitopes corresponding to mAbs 093G9 and 096B7

To account for the neutralization activity of mAb 093G9, the epitopes corresponding to mAbs 093G9 and 096B7 were further identified through screening of a phage library displaying random C7C-mer (for 093G9) and 12-mer (for 096B7) peptides simultaneously. In the third round of selection, phages that bound with higher affinity to mAb 093G9 and to mAb 096B7 were enriched. In this selection round, sequencing of the inserts of 25 clones recovered from the wells of mAb 093G9 and 32 clones recovered from the wells of mAb 096B7 revealed their difference. The consensus motif can be summarized as L(P/L)N(W/F/C)(N/T/L)P(H/Q/T) (amino acid separated by a solidus indicated the amino acid substitutions) for 093G9 and WHTRTNDQ for 096B7, respectively (Table 2). The selected consensus motif LPNLGCP showed 100% homology to the segment of human Cyr61 VWC domain (amino acid 140–146) and TR-VTND of human Cyr61 TSP-1 region (amino acid 249–254). The binding of mAb 093G9 and mAb 096B7 to the corresponding epitopes was also confirmed with ELISA analysis (data not shown). These results further demonstrated that VWC fragment was a key domain of human Cyr61 for cancer cell proliferation, migration, and tumor invasion. Blockade of Cyr61 VWC domain by mAb 093G9 (LPNLGCP) could therefore inhibit cancer cell proliferation, migration, and invasion in vitro and in vivo. In contrast, tickling to Cyr61 TSP-1 region by 096B7 (TR-VTND) did not show any such effects.

Table 2 Identification of the epitopes recognized by anti-Cyr61 mAbs 093G9 and 096B7
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Discussion

Overexpression of Cyr61 has been reported in breast cancer patients with different clinical stage, which contribute to breast cancer tumorigenesis and cancer progression [5, 7, 14–18, 20, 29]. Cyr61-transfected MCF-7, a lower growth and migration breast cancer cell line, rendered more proliferation, migration, and invasion capability, resemble to its counterpart, MDA-MB-231 [7, 18, 20]. In this study, using MDA-MB-231 cell line model, we not only demonstrated that blockade of Cyr61 by neutralizing mAb would arrest breast cancer cell proliferation, migration, and invasion in vitro and in vivo but also identified VWC region of Cyr61 as a potential novel domain contributing to breast cancer development.

We found that Cyr61 was overexpressed among 66–100% breast cancer Chinese patients with different clinical stages and pathological types and lymph node metastasis as previous reports [15, 18, 19]. Interestingly, we also observed that Cyr61 was highly expressed in breast cancer patients with ovarian metastasis. Although lymph node metastasis is common and plays an important role in clinical diagnosis and treatment of breast cancer, overexpression of Cyr61 found in ovarian metastasis is rather important for early diagnosis and treatment for patients [29, 30]. Thus, the finding of Cyr61 overexpression in ovarian metastasis may be not only as a potential therapeutic target but also as a potential risk factor for ovarian metastasis.

Our previous study has shown that Cyr61 plays a critical role in fibroblast synoviocyte (FLS) proliferation through IL-17 pathway [8]. In breast cancer research, whether the proliferation of MDA-MB-231 could be inhibited through Cyr61-specific blockade in vivo remains unknown. In current study, using RNAi technique, we successfully arrested proliferation of MDA-MB-231 cells. Moreover, we showed that anti-Cyr61 monoclonal antibody (093G9) could significantly inhibit proliferation, migration, and invasion of MDA-MB-231 cells. Similarly, this neutralizing mAb could inhibit MDA-MB-231 tumor growth and lymph node metastasis in vivo.

It is known that there are different integrin subunits expressed on cell surface as Cyr61 receptors [1, 3, 5, 7, 8, 25, 31]. Cyr61 interacting with special integrins will result in unique biological consequence via various signaling pathways. For example, on gastric cancer cell, AGS cell line, overexpressing Cyr61 may activate NF-κB via α3/COX2 pathway [10]. On breast cancer cell line, Cyr61 interacting with integrin αvβ3 will trigger FAK-PI3 K signaling pathway to promote cells proliferation, in which AKT acts as a critical kinase. Blocking Cyr61–αvβ3 interaction will decrease phosphorylation of AKT [1, 10, 18, 32]. Nevertheless, ERK1/ERK2 MAPK is reported to involve in Cyr61-transfecting MCF-7 activation through αvβ3 receptor without change in AKT activation recently [25, 33]. However, using MDA-MB-231 cell line, we found that phosphorylated AKT and ERK1/ERK2 remarkably decreased when Cyr61 was blocked by mAb 093G9. Our study indicated that in MDA-MB-231 cells, Cyr61 could activate FAK-PI3 K and ERK1/ERK2 MAPK signaling pathways simultaneously as reported in ovarian carcinomas [34].

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent enzymes that cleave extracellular matrix (ECM) proteins during cancer invasion [35–37]. Previous reports implicated that MMP-1 was one of the most highly upregulated MMPs in breast cancer and involved in invasion and metastasis [38, 39]. Recently, MMP1 expressed by breast cancer cells is reported as a mediator of transendothelial migration, tumor seeding, and relapse to lungs and brain [31, 40, 41]. On the other hand, tumor-derived Cyr61 could induce stromal MMP-1 production and thereby mediate tumor cell migration and invasion [38]. Blockade of Cyr61 might be benefit for alleviating breast cancer invasion and metastasis. TIMP1 and TIMP2 are considered as main inhibitors of MMP-1 in breast cancer cells [35, 42–45]. In this study, using neutralizing antibody 093G9, we found that blockade of Cyr61 reduced cancer cell migration and invasion companied by TIMP1 and TIMP2 upregulated dramatically. Furthermore, in nude mice model, we found that lymph node metastasis was decreased remarkably in 093G9-administrated group. These data indicated that neutralizing Cyr61 could alleviate breast cancer cells invasion via upregulation of MMP1 inhibitors, TIMP1 and TIMP2.

Given that Cyr61 consists of four structural domains [1], reports implicated that the domain 4 of Cyr61 is important for promoting tumor cell proliferation, migration, and invasion in breast cancer research [27]. However, in current study, using Ph.D.-C7C phage library display system, we identified that peptide LPNLGCP located in domain 2, VWC domain (aa140–146), of Cyr61 was recognized by 093G9, while the epitope of mAb 096B7 was a linear consensus motif TR-VTND located in TSP-1 region (aa249–254). This demonstrated that the domain 2 of Cyr61 plays an important role in breast cancer cell tumorigenesis and cancer progression.

VWC domain, also referred to as chordin-like cysteine-rich (CR) repeats, is a common motif found in more than 500 ECM proteins [1, 23, 24, 26, 33]. In Cyr61 protein, this region with typically 70–100 amino acids in length can bind to αvβ3 and contains ten conserved cysteine residues and a pair of cysteine-containing motifs [1]. The functional study shows that VWC repeats in CCN proteins regulate bone morphogenetic proteins (BMPs) and TGF-β signaling. Some reports implicated that the domain is the one to bind integrin and thereby mediate Cyr61 function. However, no evidence proved role of this domain directly involved in breast cancer development. Our data indicated for the first time that VWC domain might play very important role in proliferation, migration, and invasion of Cyr61 high-expression breast cancer cells.

Epitope recognized by 096B7 located in the TSP-1 domain, the third domain of Cyr61, was observed in our study. The TSP-1 domain is able to bind sulphated glycoconjugates and interacts with α6β6, the same as the CT domain of Cyr61 [1]. Although it is suggested to play a role in adhesion or interactions with the ECM, the exact function of the TSP-1 domain remains unclear. Our data suggested that direct binding to this domain cannot abolish the function of Cyr61 in breast cancer cell proliferation, migration, and invasion.

Taken together, in this study, we found the inhibition of growth and lymph node metastasis of breast cancer cells by blockade circular functional epitope of VWC domain of Cyr61 using mAb 093G9, which might be a novel potential target for developing therapeutics to Cyr61 overexpressing breast cancer.