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[Cancer Research 63, 3598-3604, July 1, 2003]
© 2003 American Association for Cancer Research

Experimental Therapeutics

Prolactin Antagonist-endostatin Fusion Protein as a Targeted Dual-Functional Therapeutic Agent for Breast Cancer1

Michael T. Beck, Nian Y. Chen, Karl J. Franek and Wen Y. Chen2

Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634 [M. T. B., K. J. F., W. Y. C.]; Oncology Research Institute, Greenville Hospital System, Greenville, South Carolina 29605 [M. T. B., K. J. F., W. Y. C.]; and Department of Biology, Converse College, Spartanburg, South Carolina 29302 [N. Y. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In previous studies (Chen, W. Y. et al., Clin. Cancer Res., 5:3583–3593, 1999; Chen, N Y. et al., Int. J. Oncol., 20:813–818, 2002), we have demonstrated the ability of the human prolactin (hPRL) antagonist, G129R, to inhibit human breast cancer cell proliferation in vitro and to slow the growth rate of tumors in mice. We further revealed that the possible mechanisms of G129R antitumor effects act through the induction of apoptosis via the regulation of bcl-2 gene expression. It has been established that to sustain tumor growth, it is necessary for the development of a network of blood vessels to bring in nutrients, a process called angiogenesis. The disruption of angiogenesis has been proven to be an effective strategy to cause regression of certain tumors. One of the best-studied angiogenesis inhibitors is endostatin, which acts through the inhibition of endothelial cells. In this study, we combine the anti-breast tumor effects of G129R and the antiangiogenic effects of endostatin by creating a novel fusion protein (G129R-endostatin) specifically for breast cancer therapy. The data presented here demonstrated that this novel fusion protein was able to bind to the PRL receptor (PRLR) on T-47D human breast cancer cells and inhibit the signal transduction induced by PRL. At the same time, G129R-endostatin inhibited human umbilical vein endothelial cell (HUVEC) proliferation and disrupted the formation of endothelial tube structures with potency similar to that of endostatin. More importantly, the therapeutic efficacy of G129R-endostatin was confirmed using a mouse breast cancer cell line 4T1 in vivo. G129R-endostatin has a significantly prolonged serum half-life as compared with that of G129R or endostatin alone, and exhibited greater tumor inhibitory effects than G129R and endostatin individually or in combination. Taken together, these data demonstrate the dual therapeutic effects of G129R-endostatin, and suggests that this fusion protein has great promise as a novel anti-breast cancer agent.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human breast cancer affects ~1.1 million women per year, and ~35% of these new cases will eventually result in death. Tumor metastasis still remains the main cause of breast cancer deaths (1) . Although with chemotherapy and radiation therapy, the prognosis has improved in some cases, these approaches may result in severe side effects. Recently, PRL3 has become one of the focal points in the investigation into the mechanism and onset of human breast cancer (2 , 3) . hPRL has been linked to breast cancer by several lines of evidence: (a) an autocrine/paracrine loop for hPRL has been demonstrated with the finding of biologically active PRL in breast cancer cells (2 , 4, 5, 6, 7) ; (b) PRLR expression levels are up-regulated in breast cancer cells and neoplastic mammary tissues (8) ; (c) there is a high breast cancer rate in transgenic mice overexpressing lactogenic hormones (9) ; and (d) inhibition of PRL activity with an antagonist inhibits the proliferation of breast cancer cells both in vitro (10) and in mouse studies (11) . In view of these studies, it is evident that PRL plays an important etiological role in breast cancer, and that the development of a PRL receptor antagonist may have potential as a therapeutic agent in treating this disease.

In previous studies, it was demonstrated that a single amino acid substitution mutation in hPRL resulted in a PRLR antagonist, G129R (5 , 10) . We have further determined that G129R inhibits human breast cancer cells through the induction of apoptosis (10) . One of the key mechanisms that controls signal transduction of breast cancer cells is the stimulation of the JAK/STAT/mitogen-activated-protein-kinase (MAPK) pathways by PRL. Our previous work has shown that G129R inhibits human breast cancer proliferation, at least in part, through the inhibition of STATs phosphorylation (12) . In addition, hPRL up-regulates the proapoptotic gene bcl-2, and G129R competitively down-regulates the bcl-2 gene expression in human breast cancer cells (13) . Furthermore, anti-breast tumor effects of G129R were confirmed by using human breast cancer xenografts in nude mice (11) . These studies provide strong evidence for the ability of G129R to inhibit human breast cancer and the potential to become a therapeutic agent for the treatment of human breast cancer.

A key factor in the maintenance of the uncontrollable growth of cancer cells is the formation of new blood vessels in the tumor mass to provide nutrients, namely tumor angiogenesis (14, 15, 16, 17) . Angiogenesis is also required for tumor metastasis to occur and, thus, the inhibition of tumor angiogenesis holds great promise as a therapeutic approach in stopping primary tumor growth and metastasis (18) . In recent years, there have been several inhibitors of angiogenesis identified including thrombospondin (TSP-1), angiostatin, protamine, and endostatin (19 , 20) . Endostatin is a Mr 20,000 COOH-terminal fragment of collagen XVIII and was first characterized in murine EOMA cells by O’Reilly et al. (21) and was later characterized in humans (22) . Endostatin is a specific inhibitor of endothelial cell proliferation and is a potent inhibitor of angiogenesis (23, 24, 25) . Although the mechanism of endostatin activity is not fully understood, the crystal structure of endostatin reveals a heparin sulfate-binding site (26) , suggesting that endostatin can inhibit such heparin-binding angiogenic factors as bFGF-2. Murine tumors that are dependent on angiogenesis for growth were successfully regressed to microscopic lesions after systemic therapy with murine endostatin (21) . Such inhibition may lead to tumor dormancy as a result of an increased level of apoptosis in endothelial cells (27) . Recently, Phase I clinical trials of endostatin have been completed, and it is currently in Phase II studies. In this study, we combined the tumor targeting and inhibitory activities of G129R with the antiangiogenic abilities of endostatin by creating a novel fusion protein (G129R-endostatin), and tested its potential dual therapeutic effects both in cell culture as well as in mouse tumor models.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Growth Conditions.
The human breast cancer cell line T-47D, mouse breast cancer cell line 4T1, and HUVECs were purchased from the American Type Culture Collection (Manassas, VA). T-47D cells were maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS (Hyclone Laboratories, Logan, UT) and 100 µg/ml gentamicin (Hyclone). 4T1 cells were maintained in RPMI 1640 supplemented with 10% FBS, 1% sodium pyruvate (Life Technologies, Inc.), 100 µg/ml gentamicin, 4.5 g/liter glucose (Sigma, St. Louis, MO), and 1 mM HEPES (Life Technologies, Inc.). HUVECs were maintained in Medium-199 supplemented with 10% FBS, 100 µg/ml gentamicin, and an EGM-2 Singelquot (Cambrex, East Rutherford, NJ). Cells were grown at 37°C in a humid atmosphere in the presence of 5% CO2.

Cloning and Expression of G129R-Endostatin Fusion Protein.
A two-step cloning procedure was used to generate a recombinant cDNA encoding G129R fused to human endostatin. Primers corresponding to G129R (5' primer; restriction site for NdeI underlined, 5'-CAT ATG TTG CCC ATC TGT CCC GGC-3', and 3' primer, restriction site for BamHI underlined, 5'-GGA TCC GCA GTT GTT GTT GTG GAT-3') were used to amplify the G129R fragment from a previous clone (10) . Primers corresponding to human endostatin (5' primer; restriction site for BamHI underlined, 5'-GGA TCC CAC AGC CAC CGC GAC TTC CAG-3', and 3' primer, restriction site XhoI with stop codon underlined, 5'-CTC GAG CTA CTT GGA GGC AGT CAT GAA GC-3') were used to amplify the gene from a Human Universal QUICK-Clone cDNA library (Clontech, Palo Alto, CA). Another 5' primer, NdeI, 5'-CAT ATG CAC AGC CAC CGC GAC TTC CAG, was used with the XhoI 3' primer for expression of human endostatin alone. All of the cDNA fragments were ligated separately into the TA cloning vector pCR2.1 (Invitrogen, Inc., Carlsbad, CA), were restriction mapped, and were sequenced. The cDNA fragments were restriction digested at the cloned restriction sites, were purified, and were ligated into the protein expression vector pET22b(+) (Novagen, Madison, WI) for the expression of G129R-endostatin and endostatin proteins. The design of the fusion protein is such that the NH2-terminal portion of endostatin is ligated to the COOH-terminal portion of G129R.

Production and Purification of Endostatin, G129R, and G129R-Endostatin Fusion Protein.
G129R was purified as described previously (10) . Endostatin and G129R-endostatin were purified according to Huang et al. (28) . Briefly, BL21 (Novagen) chemically competent cells were transformed with pET22b(+) vector encoding for endostatin, G129R, and G129R-endostatin cDNA. Bacteria were allowed to grow overnight in Luria-Bertani broth (ampicillin, 50 µg/ml) at 37°C. The next day the bacteria were induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) for 5 h to induce protein expression. Bacteria were collected and were resuspended in 100 ml of buffer A [0.1 M Tris-HCl (pH 8.0) and 5 mM EDTA], followed by incubation at room temperature for 15 min, with the addition of lysozyme at a final concentration of 50 µg/ml. The suspension was then sonicated using a 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) in the presence of 0.1% sodium deoxycholate, followed by centrifugation at 8000 x g for 10 min. The pellet was resuspended in 100 ml of buffer A containing 0.1% sodium deoxycholate. The centrifugation/resuspension procedure was repeated twice. The pellet was dissolved in 30 ml of buffer B [0.05 M Tris (pH 8.0), 1% SDS, and 1 mM DTT] and was centrifuged at 8000 x g for 10 min at 4°C. The clear supernatant obtained was then transferred to dialysis tubing with a Mr cutoff of 10,000 and was dialyzed twice in 1500 ml of buffer C [0.05 M Tris-HCl (pH 8.0) and 0.1 mM DTT] at 4°C for 4 h. The recombinant protein was then further dialyzed twice in 1500 ml of buffer D [0.05 M Tris-HCl (pH 8.0)] and twice in 1000 ml of buffer E [0.05 M Tris-HCl (pH 8.0), 0.01 mM oxidized glutathione, and 1 mM reduced glutathione] at 4°C for 4 h/dialysis cycle, respectively. A final dialysis in 0.05 M Tris-HCl (pH 8.0) was performed overnight. Both endostatin and G129R-endostatin were soluble in the dialysis buffer. The G129R protein was purified on a fast-performance liquid chromatography system (FPLC; Amersham Pharmacia, Newark, NJ) after refolding as described previously (12) . The endostatin and G129R-endostatin fusion protein preparations contain ~400 EU/mg protein and G129R preparation contains <5 EU/mg protein as tested by the Gel-Clot method (Cape Cod, Inc). The concentration of G129R, endostatin, and G129R-endostatin was determined by the Bio-Rad protein assay method (Bio-Rad, Hercules, CA) and G129R and G129R-endostatin were further verified using a hPRL IRMA kit (DPC, Inc., Los Angeles, CA). The purity of the proteins was determined on a SDS-PAGE gel stained with Coomassie Blue (Fisher Scientific).

Immunoblot Analysis.
G129R, endostatin, and G129R-endostatin were separated on a 4–15% SDS-PAGE gel. The proteins were transferred to enhanced chemiluminescence Hybond nitrocellulose (Amersham Pharmacia) at 12 W for 2 h. The nitrocellulose blot was blocked with TBS containing 0.05% Tween 20 and 5% milk (blocking buffer) for 1 h at room temperature. Blots were incubated overnight at 4°C in blocking buffer containing the appropriate antibody [rabbit antihuman endostatin, 1:200 (Oncogene Research Products, San Diego, CA); rabbit anti-hPRL antiserum, 1:1000 (Dr. A. Parlow, National Hormone and Pituitary Program, NIH, Bethesda, MD)]. The blots were washed three times, 5 min each, with TBS containing 0.05% Tween, and were incubated with the secondary antibody goat-antirabbit horseradish peroxidase (1:5000; Bio-Rad) for 2 h at room temperature with gentle agitation. Blots were washed three times, 5 min each, with TBS containing 0.05% Tween and were developed for 1 min using the ECL Western detection reagents (Amersham Pharmacia). Immunoblots were visualized using Kodak MR film (Fisher).

Radioreceptor Binding Assay.
T-47D human breast cancer cells expressing the PRL receptor were grown to confluency (~105 cells/well) in six-well tissue culture plates. Cells were starved in serum-free RPMI 1640 for 1 h, and then were incubated for 2 h at room temperature in serum-free RPMI medium containing 125I-labeled hPRL (specific activity, 40 µCI/µg; NEN Perkin-Elmer, Boston, MA) with or without various concentrations of PRL, G129R, endostatin, and G129R-endostatin. Cells were washed three times with serum-free RPMI medium and were lysed in 0.5 ml of 0.1 N NaOH/1% SDS. The bound radioactivity was determined by scintillation counting, and the percentage of specific displacement was calculated and compared among these samples.

Immunofluorescence Staining.
T-47D cells and HUVECs were maintained as described previously. Cells were passed onto Lab-Tek Chamber Slide System (Fisher) and were grown to ~70% confluency. HUVECs were cultured in low-serum medium (2% FBS), and T-47D cells were serum depleted for 30 min. Cells were treated with 10 µg/ml (435 nM) of G129R, 10 µg/ml (500 nM) of endostatin, or 20 µg/ml (476 nM) of G129R-endostatin for 30 min at 37°C. Cells were treated in their respective serum-free media, and all of the staining was performed in triplicate and repeated at least twice. After treatment, cells were washed with PBS [120 mmol NaCl; 2.7 mmol KCl; and 10 mmol phosphate buffer salts (pH 7.4)], fixed with 4% para-formaldehyde (BD Biosciences, Bedford, MA) for 25 min at 4°C, and permeabilized with 0.2% Triton X-100 in 1x PBS. Cells were incubated in blocking buffer for 30 min with 2% BSA (Fisher). Cells were incubated with the primary antibodies rabbit antihuman endostatin (Ab-2), 1:200, and mouse anti-hPRL antiserum, 1:1000, at room temperature for 2 h. After incubation, cells were washed three times with 1% BSA/PBS and subjected to secondary antibody (1:500) incubation for 2 h at room temperature using Alexa Fluor 594 goat antimouse IgG (red fluorescence) and Alexa Fluor 488 goat antirabbit IgG (green fluorescence; Molecular Probes, Inc., Eugene, OR), respectively. Cells were rinsed twice with 1% BSA/PBS and incubated with Anti-Fade equilibrium buffer (10 µl/well; Molecular Probes) for 10 min at room temperature. The chambers were then removed and cover slides were mounted for observation. All of the wells were examined under an Zeiss LSM 510 confocal microscope using 488-nm and 594-nm wavelengths. Digital photographs were taken at x450.

STAT-5 Phosphorylation Assay.
T-47D cells were grown to 80% confluency in six-well plates in RPMI 1640 containing 10% charcoal-stripped FBS. On the day of the experiment, cells were depleted for 30 min in RPMI 1640 containing 0.5% charcoal-stripped FBS. Cells were then treated for 20 min with the appropriate amount of PRL, G129R, endostatin, G129R-endostatin, or a combination treatment as indicated in Fig. 4Citation . Cells were washed with ice-cold PBS and were lysed with 200 µl of lysis buffer [50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM Na3VO4] and were incubated on an orbital shaker for 10 min at room temperature. The lysate was transferred to a sterile 1.5-ml centrifuge tube, gently passed through a 21-gauge needle six times, and then incubated on ice for 20 min. The lysate was centrifuged at 12,000 x g for 20 min at 4°C. The supernatant was removed, and 30 µl of the lysate (65–70 µg) was used for Western blotting analysis as described earlier, with the exception that anti-STAT5A + anti-STAT5B [1:4000; Upstate Biotechnology Inc. (UBI), Lake Placid, NY] or anti-phospho-STAT5 (1:5000; UBI) were substituted as the primary antibodies.



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  		Fig. 4. Inhibition of STAT-5 phosphorylation by G129R-endostatin. T-47D human breast cancer cells were treated with the indicated amounts of PRL, G129R, and G129R-endostatin (A) or a dose-dependent combination treatment (B). Total protein was extracted and analyzed on a 4–15% gradient SDS-PAGE, followed by Western blotting with antiserum against either STAT-5-phosphorylated (pStat5) or STAT-5 as indicated in the appropriate panel. A, inhibition or stimulation of STAT5 phosphorylation of T-47D cells by PRL, G129R, endostatin, and G129R-endostatin. B, dose-dependent competitive inhibition of STAT-5-phosphorylation by G129R-endostatin. T-47D cells were incubated with PRL and increasing concentrations of G129R or G129R-endostatin. STAT5 and phosphorylated-STAT5 were detected by Western blot analysis as described in the "Materials and Methods."

 
Cell Proliferation Assay.
HUVEC’s and T-47D cells were grown in their respective media free of phenol-red. Fully confluent HUVEC and T-47D cell cultures were trypsinized, and cells were resuspended in medium containing 5% FBS. Cells were then seeded into 96-well culture plates at a density of 5,000 HUVECs/well [in the presence of 2.5 ng/ml bFGF (Sigma) and 1 µg/ml heparin (Sigma)] and 15,000 T-47D cells/well. After an incubation of 24 h, various concentrations of G129R, endostatin, or G129R-endostatin were added to the appropriate well. Cells were further incubated for 72 h at 37°C in a humidified 5% CO2 incubator. The viability of the cells was determined using the MTS-PMS (CellTiter 96 Aqueous kit; Promega Corp., Madison, WI) colorimetric assay (following the manufacturer’s instructions), and absorbance at 490 nm was determined using a microplate reader (Bio-Rad). Cell survival was calculated as a percentage of the control values. All of the experiments were carried out in triplicate.

Endothelial Tube Formation Assay.
Matrigel (BD Biosciences) was added (320 µl) to each well of a 24-well plate and allowed to polymerize at room temperature for 20 min. A suspension of 30,000 HUVECs/well in 300 µl of Medium 199 containing EGM-2 without antibiotics was transferred into each well. The cells were then treated with a low (100 ng/ml) and high (1000 ng/ml) concentration of G129R (4.3 nM, 43 nM), endostatin (5 nM, 50 nM), or G129R-endostatin (2.4 nM, 24 nM). All assays were performed in triplicate and were repeated at least twice. Cells were incubated for 24–48 h at 37°C in a humidified 5% CO2 incubator were and observed using a CK2 Olympus microscope (3.3 ocular, x10 objective).

Pharmacokinetic Study.
Female BALB/c mice (Jackson Lab, Bar Harbor, ME) were used to determine the serum-effective dose of G129R-endostatin after a single i.p injection. Two hundred µg of G129R (8.7 nmol), 200 µg of G129R-endostatin (4.8 nmol), or 200 µg (10 nmol) of endostatin was injected (i.p.) into BALB/c mice (n = 4). Blood samples were obtained from each mouse at time intervals of 2, 4, 8, and 24 h by tail vein bleeding. Samples were placed on ice and immediately centrifuged for 5 min at 4°C. The serum was collected and frozen at -20°C until further use. The serum concentration of both G129R and G129R-endostatin was determined using the hPRL IRMA kit (DPC, Inc.). Endostatin serum concentration was determined using the Accucyte ELISA method (Oncogene). Area under the curve (AUC) was calculated by linear trapezoidal method from 2 to 24 h.

Antitumor Effects in Vivo.
The in vivo antitumor efficacy of G129R-endostatin was examined using a 4T1 mouse mammary xenograft in an athymic nude mouse model. Female athymic nude (nu/nu) mice (Jackson Lab) 6–8 weeks of age were randomly placed into groups of 5 mice/cage, two cages/treatment for a total of 10 mice/group. 4T1 breast cancer cells (5 x 104) were injected s.c. into the mammary fat pad of each mouse, and tumors were allowed to develop for 5 days. Once tumors were established, mice were subjected to daily i.p. injections of different agents as designed. Treatment groups were given G129R [2.5 mg (110 nmol)/kg/day], endostatin [2.5 mg (125 nmol)/kg/day], G129R-endostatin [5 mg (130 nmol)/kg/day], and a combination of G129R (2.5 mg/kg/day) and endostatin (2.5 mg/kg/day) in a volume of 100 µl. Control groups were given 100-µl injections of sterile PBS. Measurements of tumors were recorded every 5 days until it was decided that tumors were debilitating to the mice. The long axis (L) and the short axis (S) were measured, and the tumor volume (V) was calculated using the following equation:

Once final measurements were taken, the mice were sacrificed by cervical dislocation, and tumors were dissected, weighed, and flash-frozen and were stored in liquid nitrogen until analysis.

Statistical Analysis.
The results from the MTS assay and the animal studies were presented as means ± SE (error bars). Statistical analysis was performed using the program StatsDirect, version 1.9.8 (CamCode, Cambridge, England) with one-way ANOVA and a Tukey-Multiple Comparison test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of G129R-Endostatin Fusion Protein.
The recombinant fusion protein along with G129R and endostatin were purified from the inclusion bodies of Escherichia coli cells. As shown in Fig. 1ACitation , all of the recombinant proteins migrate as a single band during SDS gel electrophoresis under reduced conditions in predicted sizes (G129R-endostatin, Mr 42,000; G129R, Mr 23,000; endostatin, Mr 20,000). Western blot analysis was used to further confirm the presence of both G129R and endostatin in the G129R-endostatin fusion protein (Fig. 1B)Citation .



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  		Fig. 1. Production and expression of G129R-endostatin. G129R and endostatin were cloned into the pET22b(+) expression vector. A, SDS-PAGE analysis of G129R-endostatin stained with Coomassie Blue. At left, molecular weight markers (kDa) in thousands. G129R migrates at Mr 23,000 (Lane 1) and endostatin at Mr ~20,000 (Lane 2). G129R-endostatin migrated at Mr ~43,000 (Lane 3). B, Western blot analysis of G129R-endostatin. Lanes 1 and 4, G129R; Lanes 2 and 5, endostatin; Lanes 3 and 6, G129R-endostatin. The left blot, Lanes 1–3, was incubated with a polyclonal rabbit anti-hPRL antibody and the right blot, Lanes 4–6, was incubated with a polyclonal rabbit antiendostatin antibody. A goat antirabbit IgG horseradish peroxidase conjugate was used as secondary antibody and was detected with ECL.

 
G129R-Endostatin Binds to Both Human Breast Cancer and Endothelial Cells.
The ability of G129R-endostatin to directly bind to the PRLR on the human breast cancer cell line T-47D was demonstrated using a radioreceptor binding assay (Fig. 2)Citation . It was determined that PRL, G129R, and G129R-endostatin all competitively displaced the 125I-labeled hPRL from the PRLR on T-47D cells with similar affinity, whereas endostatin did not, suggesting that G129R-endostatin retained its ability to recognize PRLR.



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  		Fig. 2. Binding ability of G129R-endostatin to the PRL receptor on human breast cancer cells. The concentrations of the treatments are given on a log scale. The data are represented as the percentage of the displacement of 125I-labeled hPRL (specific activity, 40 µCI/µg) compared with the total binding of each protein to human breast cancer cell line T-47D. The data represents the mean ± SD of three experiments.

 
An immunofluorescence assay was used to determine whether G129R-endostatin can bind to both breast cancer and endothelial cells (Fig. 3)Citation . HUVEC and T-47D cells were treated with G129R, endostatin, or G129R-endostatin and were stained with protein-specific primary antibodies. Fluorescent secondary antibodies were used to distinguish G129R (Alexa Flour 594, Red) and endostatin (Alexa Flour 488, Green). Fig. 3, A and BCitation represent the untreated HUVECs and T-47D cells as controls. As shown in Fig. 3, C and DCitation , G129R-endostatin bound to HUVEC and T-47D cells, respectively. This is demonstrated by the fluorescence of both the endostatin antibody (green) and the PRL antibody (red) in the same field of view. Endostatin bound to HUVECs (Fig. 3E)Citation and bound to what appears to be the ECM of T-47D cells with a scattered staining pattern (Fig. 3F)Citation . In contrast, G129R bound only to T-47D cells (Fig. 3H)Citation , but it did not bind to HUVECs (Fig. 3G)Citation . The distinct pattern of staining of G129R and endostatin is notable. G129R and G129R-endostatin treatments revealed a clear cellular staining pattern in T-47D cells (Fig. 3, D and H)Citation , whereas endostatin-treated cells demonstrated a scattered staining pattern in both HUVECs and T-47D cells (Fig. 3, C, E, and F)Citation . Because G129R did not bind to HUVECs caused by the lack of PRLR on these cells (Fig. 3G)Citation , the staining of HUVECs by G129R-endostatin (Fig. 3C)Citation was most likely caused by the binding of the endostatin portion of the fusion protein.



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  		Fig. 3. Immunofluorescence staining of HUVECs and T-47D cells. C (HUVECs) and D (T-47D) represent cells treated with G129R-endostatin and stained with anti-hPRL and anti-human endostatin. C and D are boxed to represent the same field of view. Both E (HUVEC) and F (T-47D) represent cells treated with endostatin and G129R and stained with antihuman endostatin. Both HUVECs (G) and T-47D (H) cells were treated with endostatin and G129R and stained with anti-hPRL. Negative controls of HUVECs and T-47D cells were presented as A and B, respectively. The secondary antibodies used were Alexa Fluor 594 goat antimouse IgG (red fluorescence, PRL) and Alexa Fluor 488 goat antirabbit IgG (green fluorescence, endostatin), respectively, for each primary antibody. Digital photographs were taken at x450.

 
G129R-Endostatin Inhibits STAT5 Phosphorylation in T-47D Human Breast Cancer Cells.
STAT5 phosphorylation is one indicator of PRL-mediated signal transduction in mammary cells, and we have used this feature as a measure of the antagonistic effects of G129R and its variants (12) . The status of STAT5 phosphorylation was examined after treatment of T-47D cells with PRL, G129R, endostatin, and G129R-endostatin. As shown in Fig. 4ACitation , PRL (100 ng/ml, 4.3 nM) induced phosphorylation of STAT5 (pSTAT5), whereas G129R, endostatin, and G129R-endostatin, as expected, lacked the ability to induce STAT5 phosphorylation. A dose-dependent competitive inhibition of PRL-induced STAT5 phosphorylation was observed for G129R and G129R-endostatin (Fig. 4B)Citation . G129R and G129R-endostatin exhibited similar potency in the inhibition of STAT5 phosphorylation. This demonstrates that the G129R portion of G129R-endostatin retained its antagonistic effects toward PRLR.

G129R-Endostatin Inhibits the Proliferation of Human Endothelial and Human Breast Cancer Cells.
Cell proliferation assays were carried out to examine the dual effects of G129R-endostatin in inhibiting the proliferation of both HUVECs and T-47D cells. G129R-endostatin was revealed to be as effective as endostatin in inhibiting the proliferation of HUVECs in a dose-dependent manner (Fig. 5A)Citation . The EC50 of G129R-endostatin (12 nM) was approximately one-half that of endostatin (25 nM; ~500 ng/ml; Fig. 5ACitation ). G129R had no effect on HUVEC proliferation, suggesting that the inhibitory effect of G129R-endostatin was caused by the endostatin domain of the fusion protein. Conversely, G129R-endostatin (EC50, 18 nM) exhibited antiproliferative effects on T-47D human breast cancer cells similar to that of G129R (EC50, 32 nM; ~750 ng/ml; Fig. 5BCitation ). As expected, endostatin had no effect on the proliferation of T-47D cells. Overall, G129R-endostatin was effective in inhibiting T-47D and HUVEC growth at molar concentrations much lower than those of G129R or endostatin, respectively.



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  		Fig. 5. Breast cancer and endothelial cell proliferation assay. Purified human endostatin, G129R-endostatin, and G129R were tested for their antiproliferative ability using HUVECs (A) and T-47D cells (B). Viability of cells was determined by the colorimetric MTS-PMS assay (Promega). Data are represented by the percentage of viable cells after treatments. A, ability of endostatin and G129R-endostatin to inhibit bFGF-induced endothelial cell proliferation using G129R as the control. B, effects of G129R and G129R-endostatin to inhibit the proliferation of human breast cancer cell line T-47D using endostatin as the control. Each experiment was carried out in triplicate, and the data are represented as the mean ± SE of three experiments.

 
G129R-Endostatin Fusion Protein Disrupts the Formation of Endothelial Tubes.
An endothelial tube formation assay was used to further confirm the antiangiogenic activity of G129R-endostatin. In this experiment, the use of Matrigel permits the growth and differentiation of endothelial cells into tubal structures that are reminiscent of blood vessels. Prominent tubal structures were observed in the control cells (Fig. 6)Citation . At low concentrations (100 ng/ml; Fig. 6Citation , left column) both endostatin and G129R-endostatin began to disrupt the formation of the tubes, indicated by the arrows. At high concentrations (1,000 ng/ml; Fig. 6Citation , right column), both endostatin and G129R-endostatin treatments eliminated the tubal structures, and the cells appeared to be dying. G129R treatment, serving as a negative control in this experiment, had no obvious effects on endothelial tube formation.



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  		Fig. 6. The effect of G129R-endostatin on the three-dimensional structure of endothelial tubes. HUVECs (25,000 cells/well) in EGM-2 medium without antibiotic were plated onto Matrigel basement membrane-coated wells and were evaluated for their ability to form tubal structures similar to that of blood vessels. A low (100 ng/ml) and high (1000 ng/ml) concentration was used for endostatin, G129R-endostatin, and G129R. Each treatment was performed in triplicate. Untreated (Control) cells were processed similar to cells receiving drug treatment. Cells were viewed with a microscope and pictures were taken at x10.

 
Pharmacokinetic Comparison of G129R, Endostatin, and G129R-Endostatin Fusion Protein.
It has been demonstrated that increasing the size of a protein may increase its half-life (29) . The relatively short serum half-life of G129R and endostatin present a considerable challenge to the clinical use of these potential therapeutic agents. To examine whether the pharmacokinetics of G129R-endostatin were improved compared with G129R or endostatin alone, the AUC values of G129R, G129R-endostatin and endostatin were determined and compared after single i.p. injection (Fig. 7)Citation . The AUC was calculated for 200 µg of each protein based on the plot in Fig. 7Citation . The AUC of G129R-endostatin is 7.3 times that of G129R and ~10 times that of endostatin. In addition, if taking into consideration the relative molar amounts of each protein injected (4.8 nmol of G129R-endostatin, as compared with 8.7 nmol of G129R or 10 nmol of endostatin was used in each experiment), the effective serum concentration of G129R-endostatin was found to be 13 times that of G129R and 21 times of equimolar amounts of endostatin. Thus, G129R-endostatin exhibits a higher effective serum concentration (and thus a longer serum half-life) than do G129R and endostatin.



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  		Fig. 7. Pharmacokinetic analysis of G129R-endostatin in BALB/c mice. Female BALB/c mice (n = 4) were given i.p. injections of G129R-endostatin (200 µg), G129R (200 µg), or endostatin (200 µg) and serum samples were collected by tail vein bleeding at the indicated time intervals. The serum concentration of G129R and G129R-endostatin was determined using the hPRL IRMA kit (DPC, Inc.). The serum concentration of endostatin was determined using the Accucyte ELISA protocol (Oncogene). The area under the curve (AUC) was determined for each protein. The AUC of G129R-endostatin is 7.3 times that of G129R. When adjusted for the relative molar amounts of each protein injected, the effective serum concentration of G129R-endostatin was found to be 13.1 times that of G129R for equimolar amounts of protein. The AUC for G129R-endostatin was 10-fold greater than the AUC for endostatin; this value was 21 for equimolar amounts of protein.

 
G129R-Endostatin Fusion Protein Inhibits the Growth Rate of Breast Cancer Xenografts in Nude Mice.
To test the efficacy of G129R-endostatin in inhibiting breast cancer, we used an aggressive murine breast cancer cell line, 4T1, in a nude mouse model. Fifty female athymic nude mice that were given injections of 4T1 cells (5 x 104) s.c. into the mammary fat pad were randomly divided into five groups. Postinoculation starting on day 5, G129R, endostatin, G129R-endostatin, and a combination treatment of G129R and endostatin were administered daily (i.p.). The control group was treated with 100 µl of PBS. Compared with control mouse group, all four of the treatments caused a significant reduction in tumor volume (P < 0.0001). Among the four treatment groups, G129R-endostatin (807 ± 235 mm3) demonstrated the best inhibitory effects on 4T1 tumor growth and exhibited a statistically significant decrease in final tumor volume, compared with the control (2851 ± 305 mm3; P < 0.001), G129R (1897 ± 194 mm3; P < 0.001), endostatin (1271 ± 142 mm3; P < 0.001), and the combination treatment (1399 ± 147 mm3; P = 0.0016) groups (Fig. 8A)Citation . Similarly, all of the treatments caused significant reduction in the final tumor weights compared with the control group (1970 ± 410 mg): G129R-endostatin (841 ± 121 mg; P < 0.001); G129R (1409 ± 265 mg; P < 0.001), endostatin (1159 ± 170 mg; P < 0.01), and the combination of G129R and endostatin (1149 ± 195 mg; P < 0.001; Fig. 8BCitation ). G129R-endostatin treatment resulted in lower tumor weights than the other treatments in general. However, although this decrease was statistically significant compared with G129R (P = 0.0004), it was not significantly greater than that brought about by endostatin (P = 0.0936) or endostatin and G129R in combination (P = 0.1065).



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  		Fig. 8. In vivo analysis of human breast cancer inhibition using G129R-endostatin. Fifty athymic nude mice per group were inoculated (s.c.) with 5 x 104 4T1 cells. Tumors were allowed to establish for 5 days. Mice were randomized into five groups of 10 and were given injections of G129R (2.5 mg/kg/mouse), endostatin (2.5 mg/kg/mouse), G129R-endostatin (5 mg/kg/mouse), the combination of G129R (2.5 mg/kg/mouse) and endostatin (2.5 mg/kg/mouse), or 100 µl of sterile PBS for 35 consecutive days. A, tumor volume was determined every 5 days posttreatment by measuring the short axis (S) and the long axis (L) of the tumors and was calculated using the equation: [S2 x L]/2. Treatments of G129R-endostatin, G129R, endostatin, and G129R and endostatin in combination caused significant tumor reduction compared with the control group (P < 0.005). B, once the final tumor volume was measured, the tumors were removed and weighed. Values are represented as mean ± SE for each group (n = 10). All of the treatments caused significant reduction in the final tumor weights compared with the control group: G129R-endostatin (P < 0.001); G129R (P < 0.001), endostatin (P < 0.01), and the combination of G129R and endostatin (P < 0.001; B). In addition, G129R-endostatin-induced decrease in tumor weight was significantly greater than in G129R-treated mice (P = 0.0004), but not the endostatin (P = 0.0936) or endostatin and G129R combination (P = 0.1065) groups.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Angiogenesis is the process of growth of new capillaries from preexisting blood vessels and is a crucial element for tumor sustenance (13) . The switch of angiogenic phenotype in a tissue is dependent on the local balance between angiogenic factors and inhibitors (17) . Of the many angiogenesis inhibitors that have been investigated and considered for potential cancer therapy, endostatin is one of the most potent and shows promise in inhibiting tumor growth in animals and in clinical trials (19) .

The underlying molecular mechanisms of antiangiogenic activity of endostatin are not fully understood, although several recent studies have begun to shed light on the mode of action of endostatin. Endostatin induces apoptosis causing G1 arrest of endothelial cells through the inhibition of cyclin D1 (30) and may interrupt the Wnt signaling pathway, which is involved in cellular development (31) . There is evidence that endostatin blocks the binding of vascular endothelial growth factor to endothelial cells (32) and inhibits the activation and catalytic activity of matrix metalloproteinases (33) . Taken together, these studies suggest that the antitumor effects of endostatin are attributable to its specificity for endothelial cell proliferation rather than the direct inhibition of tumor cell growth (19) . Successful attempts have been made to target endostatin to cancers of the breast and other tissues. For example, liposomes complexed with plasmids that encode endostatin inhibit breast tumor growth in mice when injected directly into tumors (34) . Adenovirus-mediated systemic gene transfer of endostatin demonstrated significant reduction of tumor growth and inhibition of micrometastases in a mouse model (35) . Together, these studies indicate that targeting endostatin directly to the tumor mass may improve the chance of tumor regression.

In view of the important role that PRL plays in breast cancer cell survival, the PRL antagonist, G129R, has demonstrated great potential as an antitumor agent. G129R inhibits breast cancer cell proliferation through the induction of apoptosis (10) , in part, through the inhibition of bcl-2 gene expression (13) . Furthermore, G129R inhibits the growth of both T-47D and MCF-7 human breast cancer xenografts in nude mice (11) . We have taken advantage of the ability of G129R to bind PRLR by designing targeted antitumor therapeutic agents. In this study, we genetically combined two proven effective anticancer agents that act via different mechanisms to create a novel bifunctional fusion protein, G129R-endostatin. We reasoned that a fusion protein consisting of G129R and endostatin would be targeted to breast cancer cells, inhibit tumor cell proliferation, and inhibit angiogenesis, which is required for proper development of the vascular network at the tumor site.

For endostatin to exert its antiangiogenic effects on the breast tumor microenvironment, both the G129R and the endostatin domains of G129R-endostatin fusion protein must recognize and bind receptors on breast cancer cells and endothelial cells, respectively. The specific binding of G129R-endostatin to the PRLR on breast cancer cells and to HUVECs was demonstrated by a radioreceptor binding assay and immunofluorescence/confocal microscopy. The binding affinity of G129R-endostatin to PRLR was similar to that of PRL and G129R. Thus, each portion of the fusion retained the ability to recognize its cognate receptor. The dual binding ability of the fusion protein was illustrated by dual immunofluorescence staining of both G129R and endostatin portions of G129R-endostatin. The binding pattern of endostatin to what appears to be the ECM in cultures of T-47D cells is interesting. The precise receptors/ligands to which endostatin binds have not been fully determined, and it is possible that, in the absence of preferred cell surface receptors on T-47D cells, endostatin associates with one or more ECM proteins. Because G129R itself has a high affinity for T-47D breast cancer cells, the G129R-endostatin fusion protein binds preferentially to these cells. Although the fusion protein binds to both breast cancer cells (T-47D) and endothelial cells via the appropriate domains, the individual domains of the fusion protein may not necessarily exhibit similar affinities for their respective ligands; the affinity of G129R for the PRLR may be greater than that of endostatin for its ligand(s) in the ECM. This may prove to be important in future clinical applications in which preferential localization of G129R-endostatin to breast tumor tissue, instead of to vascular tissue in general, is essential.

Drug efficacy is, in part, affected by its serum half-life, a property that can be improved by increasing the size of a given molecule or protein (36) . A potential limitation of the use of G129R and endostatin in cancer treatment is their relatively short serum-half-lives (29) . One incentive to generate G129R-based fusion proteins for cancer therapy was to increase the serum half-life of G129R by increasing its size, a strategy that we used to generate a G129R fusion with interleukin 2 (G129R-interleukin 2; 29 ). In previous studies, G129R inhibited breast cancer xenografts at a dose of 5 mg (220 nmol)/kg/day (11) , whereas inhibition of tumor growth, and an increase in serum half-life could be achieved by increasing endostatin to 20 mg (1 µmol)/kg/day (19 , 21) . The serum half-life of endostatin in mice has been found to be ~5 h (37) . We have increased the serum half-life of G129R by mixing it with Matrigel or incorporating it into slow-releasing pellets (11) , however, these methods currently are unsuitable for clinical studies. In this study, we demonstrate that one advantage of generating novel fusion proteins as therapeutics is that, along with the increased molecular size of the fusion protein, their serum half-lives are usually greatly extended. The effective serum concentration of G129R-endostatin is maintained over 24 h as shown in Fig. 7Citation ; this is significantly longer than that of either endostatin or G129R. We believe that this feature should contribute significantly in enhancing the antitumor effects of G129R-endostatin, especially in a clinical setting.

In summary, we have created a novel fusion protein, G129R-endostatin, consisting of the PRL antagonist G129R and the antiangiogenic protein endostatin. The fusion protein is a bifunctional protein that exhibits characteristics of G129R (the inhibition of breast cancer cell proliferation) and endostatin (inhibition of endothelial cell proliferation and development). More importantly, G129R-endostatin inhibits tumor growth at a dose much lower (5 mg/kg/day) than that reported for previous endostatin treatments (20 mg/kg/day). Given its bifunctional nature, G129R-endostatin, could become a potential therapeutic agent for the treatment of human breast cancer. Additional studies of the in vivo efficacy of G129R-endostatin will support its potential benefit in clinical application. The shortcomings of endostatin Phase II/III clinical trials may be ameliorated by a strategy, as described here, which increases the effective serum concentration of endostatin and targets it directly to the tumor site.


   ACKNOWLEDGMENTS
 
We thank Jang Pyo Park, Susan Peirce, John Langenheim, Michele Scotti, and Dr. Thomas Wagner for their contributions to this project. We would also like to thank Eric Holle and Debra Cooper for their outstanding technical support for the mouse studies. Our appreciation goes to Brenda Welter and Dr. Lesly A. Temesvari for sharing their expertise in confocal microscopy.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the Endowment Fund of the Greenville Hospital System, United States Army Medical Research Command Grant DAMD17-99-1-9129), and NIH/National Cancer Institute Grant 1R21CA87093. Back

2 To whom requests for reprints should be addressed, at Oncology Research Institute, Greenville Hospital System, Greenville, South Carolina 29605. Phone: (864) 455-1457; Fax: (864) 455-1567; E-mail: wchen{at}ghs.org Back

3 The abbreviations used are: PRL, prolactin; hPRL, human PRL; PRLR, hPRL receptor; bFGF, basic fibroblast growth factor; HUVEC, human umbilical vein endothelial cell; FBS, fetal bovine serum; IRMA, immunoradiometric assay; TBS, Tris-buffered saline; ECM, extracellular matrix. Back

Received 10/16/02. Accepted 4/28/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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