Expression of angiostatin cDNA in a murine renal cell carcinoma suppresses tumor growth in vivo

BASIC SCIENCE

EXPRESSION OF ANGIOSTATIN cDNA IN A MURINE RENAL CELL CARCINOMA SUPPRESSES TUMOR GROWTH IN VIVO TOMOHARU FUKUMORI, MASA-AKI NISHITANI, TAKUSHI NARODA, TOMOICHIRO ONISHI, NATSUO OKA, HIRO-OMI KANAYAMA, AND SUSUMU KAGAWA

ABSTRACT Objectives. To investigate the effectiveness of angiostatin gene therapy for renal cancer using a mouse model. The generally poor prognosis of advanced renal cancer indicates the need for new therapeutic modalities. The dependency of solid tumor growth on angiogenesis suggests that antiangiogenic therapy would be effective against renal cell carcinoma, which is generally a hypervascular tumor. Methods. Murine renal cancer cells (Renca) transfected with murine angiostatin cDNA (AST-Renca) were subcutaneously implanted in BALB/c mice. Subsequently, the macroscopic appearance and volume of tumors were evaluated once per week. Renca cells transfected with empty plasmid DNA (mock-Renca) were used as a control. In addition, histologic sections of tumor were analyzed for neovascularization on the basis of an immunohistochemical analysis for CD31. The antitumor effect of AST-Renca on a parental Renca tumor at a distant site was also evaluated. Results. The mean volume of AST-Renca tumors was significantly less than that of the control vectortransfected tumors 3 weeks after implantation. In the cell proliferation assay, the expression of angiostatin did not inhibit the proliferation of Renca cells in vitro. Immunohistochemical analysis of neovascularization by staining with anti-CD31 antibody revealed that angiostatin suppressed tumor vessel formation. Moreover, implantation of AST-Renca inhibited the growth of parental Renca implanted simultaneously at a distant site. Conclusions. Expression of an angiostatin transgene can suppress the growth of murine renal cancer through the inhibition of tumor-induced angiogenesis. Angiostatin gene therapy may be effective against renal cancer. UROLOGY 59: 973–977, 2002. © 2002, Elsevier Science Inc.

R

enal cortical tumors are a complex family of tumor of which the conventional clear cell type is the most virulent member and accounts for 65% of all tumors. This tumor is characterized by a lack of early warning signs, resulting in a high population of patients with metastasis at diagnosis.1–3 The outlook for patients with distant metastasis is poor, because no adequate, effective treatment is available for these patients. Prior studies have shown that clear cell carcinoma is resistant to cytotoxic chemotherapy and radiotherapy. Only imThis work was supported in part by a Grant-in-Aid for Scientific Research (C) (grant 11671502) from the Japan Society for the Promotion of Science. From the Department of Urology, University of Tokushima School of Medicine, Tokushima, Japan Reprint requests: Susumu Kagawa, M.D., Department of Urology, University of Tokushima School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan Submitted: September 25, 2001, accepted (with revisions): February 4, 2002 © 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED

munotherapy with interferon and/or interleukin-2 achieves responses in 10% to 20% of patients with distant metastasis.1 New therapeutic modalities against advanced renal cell carcinoma are needed. Recent studies have shown that angiogenesis plays an important role in the growth, progression, and metastasis of solid tumors, including renal cell carcinoma.4 – 8 Renal cell carcinomas are generally hypervascular tumors. Moreover, previous studies have shown a relationship between vascularity and tumor aggressiveness4,7; therefore, modulation of its neovascularization by antiangiogenic gene therapy may be a novel strategy for the treatment of renal cell carcinoma, although antiangiogenic therapy seems to be effective for both hypervascular and hypovascular tumors in animal models.9 Angiostatin is one of the endogenous inhibitors of angiogenesis generated by the proteolytic cleavage of plasminogen.10 It has been shown to inhibit cancer growth in mice,11–13 apparently by preventing the formation of new blood vessels.14,15 The 0090-4295/02/$22.00 PII S0090-4295(02)01615-1 973

activity of angiostatin transgenes has recently been tested in several tumor models.16 –21 However, the antiangiogenic effect of angiostatin on renal cancer has not yet been fully clarified. To our knowledge, the present study is the first to investigate angiostatin gene therapy for renal cancer. To examine the potential role of angiostatin in gene therapy for renal cancer, we transfected cDNA coding for mouse angiostatin into murine renal cancer cells, implanted these cells into mice, and analyzed the antitumor and antiangiogenic effects in vivo. MATERIAL AND METHODS CELLS AND TRANSFECTION The murine renal cell carcinoma cell line, Renca, was maintained in RPMI-1640 medium supplemented with 10% heatinactivated fetal bovine serum. For transfection, recombinant plasmid DNA or empty plasmid DNA was introduced into Renca cells by LipofectAMINE (Invitrogen, Carlsbad, Calif). Transfected cells were selected with 1 mg/mL Geneticin (Invitrogen) in RPMI-1640 containing 10% fetal bovine serum for 2 weeks. G-418-resistant single cell clones were picked and expanded.

CONSTRUCTION AND TRANSFECTION OF THE MURINE ANGIOSTATIN CDNA A cDNA coding for mouse angiostatin was synthesized by polymerase chain reaction using the mouse plasminogen cDNA: forward primer, 5⬘-GGATTGGATCCTTGTTGGCCAGTCCCA-ACATGGACCATAAGGAAG; reverse primer, 5⬘-CCGAGGAATTCTTACTAGAGG-CTAGCGTAATCCGGAACATCGTATGGGTATCCTGTCTCTGAGCACCGCTTCAG. The final angiostatin cDNA-encoded amino acid residues 1-458 of plasminogen was flanked by BamH1-EcoRI restriction site linkers and encoded a signal sequence, the preactivation peptide, and kringle domains 1– 4. An antigenic epitope tag derived from influenza hemagglutinin (HA) was fused in frame to the COOH terminus of kringle 4. The polymerase chain reaction-amplified cDNA fragments were cloned into the cytomegalovirus-derived expression vector, pcDNA3.1 (Invitrogen).12,16

WESTERN IMMUNOBLOTTING Western blots were performed as described previously.16 Angiostatin and mock-transfected confluent cells were incubated for 4 days at 37°C. Then, 5 mL of each individual clone’s conditioned medium was harvested and incubated overnight at 4°C with 300 ␮L of 50% lysine-Sepharose (Pharmacia Biotech, Uppsala, Sweden) in 50 mM Tris-HCl, pH 8.0. The Sepharose beads were washed three times with 50 mM TrisHCl, pH 8.0. The bound proteins were released by adding 50 ␮L of sodium dodecyl sulfate (SDS) sample buffer and then heating samples at 95°C for 5 minutes. After separation on a 12% SDS-polyacrylamide gel, proteins were transferred onto a nitrocellulose membrane. The membrane was blocked for 30 minutes in a blocking buffer (0.1% Tween 20, 5% skim milk, 150 mM NaCl, and 20 nM Tris-HCl, pH 7.5), and incubated with a mouse monoclonal anti-HA antibody (Boehringer Mannheim, Indianapolis, Ind). After washing, the membrane was incubated with a horseradish peroxidase-coupled secondary antibody (Amersham Pharmacia Biotech, UK). Blots were developed using an ECL system (Amersham Pharmacia Biotech). 974

ANIMAL STUDIES

A total of 5 ⫻ 105 Renca cells transfected with murine angiostatin cDNA (AST-Renca) or empty plasmid DNA (mockRenca) were subcutaneously implanted into the abdominal area of BALB/c mice, and the growth of the tumors was monitored two times per week using tissue calipers. Tumor volume was calculated as ␲/6 ⫻ [(a ⫻ b)1/2]3, where a and b are two perpendicular major diameters. This experiment was performed in duplicate, with at least 5 mice studied for each treatment condition (AST-Renca and mock-Renca). To evaluate the antitumor effect of AST-Renca on a tumor at a distant site, 105 parental Renca cells were subcutaneously implanted into the breast area of BALB/c mice that were subcutaneously inoculated with 5 ⫻ 105 AST-Renca cells, mock-Renca cells, or no cells in the abdominal area, and the growth of the parental Renca tumors was monitored two times per week. This experiment was also performed in duplicate, with 10 mice studied for each treatment condition (AST-Renca, mockRenca, and no cells).

CELL PROLIFERATION ASSAY To assess the proliferation of AST-Renca and mock-Renca in vitro, 104 tumor cells of each selected clone and parental cells were seeded with 10% fetal calf serum-RPMI onto 24-well culture plates in triplicate. Cells were dispersed with trypsin, resuspended in phosphate-buffered saline, and counted every 24 hours by a Coulter counter for 5 days.

HISTOLOGIC STUDIES Four weeks after implantation, the tumors were surgically removed. Tumor tissue samples were fixed in alcohol formalin acetic acid, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. After routine histologic examination, samples were deparaffinized with xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by immersing sections in 0.3% hydrogen peroxide in methanol for 30 minutes. They were then treated with 0.2 mg/mL proteinase K for 20 minutes at 37°C, incubated with normal rabbit serum at room temperature for 30 minutes, and incubated overnight at 4°C with anti-CD31 antibody at a dilution of 1:200 (5 ␮g/mL). Subsequently, biotinylated secondary antibody (LSAB-Kit, Dako, Carpinteria, Calif) was applied to the sections for 30 minutes. Streptavidin-biotinylated peroxidase complex (LSAB-Kit, Dako) was then applied, and sections were incubated with the chromogen diaminobenzidine (Wako, Osaka, Japan). Slides were counterstained with hematoxylin.

RESULTS DETECTION OF ANGIOSTATIN IN CONDITIONED MEDIA FROM STABLE CLONES To study the constitutive expression of HAtagged angiostatin, stable cell clone culture medium was harvested after 4 days of incubation. Immunoblotting experiments with the anti-HA antibodies detected a 58-kDa protein (Fig. 1), the expected full-length angiostatin, in the conditioned media of the angiostatin-transfected clone. It should be emphasized that the molecular mass of recombinant mouse angiostatin was higher than that of the originally reported angiostatin fragment (38 kDa). The increased molecular mass of recombinant angiostatin could have been due to the presUROLOGY 59 (6), 2002

FIGURE 1. Detection of angiostatin proteins by Western blotting. Renca cells were transfected with pcDNAAST (lane 1) or pcDNA (lane 2). Culture supernatants were prepared (see Material and Methods section) and subjected to Western blot analysis using anti-HA antibody. The immunoblots showed that the expected 58kDa protein was present in the stable cell line culture medium.

ence of the preactivation peptide and an HA tag in our construct.16 EXPRESSION OF ANGIOSTATIN INHIBITS MURINE RENAL CANCER GROWTH IN VIVO BUT NOT IN VITRO We tested the antitumor effect of expression of murine angiostatin against murine renal cancer, Renca, in vivo. Significant inhibition of primary tumor growth was observed in angiostatin-transfected tumors 3 weeks after implantation (ASTRenca, 292.2 ⫾ 160.1 mm3 versus mock-Renca, 577.6 ⫾ 266.3 mm3, P ⬍0.05). Five weeks after implantation, the difference was more significant (AST-Renca, 451.3 ⫾ 105.5 mm3 versus mockRenca, 1695 ⫾ 462 mm3, P ⫽ 0.005; Fig. 2A). To investigate further whether suppression of tumor growth in the angiostatin-transfected cells was due to an antiangiogenic effect or direct antitumor activity, cell growth was tested in vitro. ASTRenca, mock-Renca, and parental Renca cells were seeded in 96-well plates, and cell growth was determined using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The three cell types exhibited the same growth rate (data not shown). This result indicates that the expression of angiostatin does not directly inhibit the growth of Renca cells. Furthermore, we evaluated the antitumor effect of AST-Renca cells on a tumor at a distant site. We found that subcutaneous inoculation of ASTUROLOGY 59 (6), 2002

FIGURE 2. (A) Inhibition of tumor growth by expression of angiostatin. Significant inhibition of tumor growth was observed in angiostatin-transfected tumors 3 weeks after implantation. Mock ⫽ mock-transfected stable cell clone; AST ⫽ pcDNA-AST-transfected stable cell clone. (B) Inhibition of tumor growth at a distant site. Significant inhibition of tumor growth at a distant site by angiostatin-transfected tumors was observed 21 days after implantation. Mock ⫽ mock-transfected stable cell clone; AST ⫽ pcDNA-AST-transfected stable cell clone.

Renca cells into the abdominal area significantly inhibited the growth of parental Renca cells subcutaneously implanted into the breast area, as revealed by examination of the parental cell tumor 3 weeks after implantation (Fig. 2B). ANGIOSTATIN INHIBITS NEOVASCULARIZATION OF MURINE RENAL CANCER Histopathologic findings of hematoxylin-eosin staining showed that the size of the blood vessels in the tumor tissue obtained from the angiostatintransfected clones was significantly less than that of the controls (Fig. 3A,B). Immunohistochemical analysis of neovascularization by staining with the anti-CD31 antibody also revealed that the expression of angiostatin inhibited tumor vessel enlargement (Fig. 3C,D), although no significant difference was found between the microvessel density of the angiostatin-treated group and that of the control group (data not shown). These results suggest that the mechanism of the tumor growth inhibition 975

FIGURE 3. Histologic findings. Angiostatin suppressed vessel formation and vascular endothelial cell proliferation. (A) Mock-transfected tumor cells (hematoxylin-eosin stain, original magnification ⫻100), (B) angiostatin-transfected tumor cells (hematoxylin-eosin stain, original magnification ⫻100), (C) mock-transfected tumor cells (antiCD31 antibody stain, original magnification ⫻400), and (D) angiostatin-transfected tumor cells (anti-CD31 antibody stain, original magnification ⫻400).

observed in this study involves suppression of tumor angiogenesis rather than a primary antitumor effect of angiostatin. COMMENT Despite advances in multimodality therapy, the 5-year survival rate for advanced renal cancer is less than 10%, mainly owing to the failure of systemic therapy to control disseminated disease.2 Antiangiogenic therapies are attractive as potential treatments for renal cancer because previous studies have shown a relationship between vascularity and tumor aggressiveness.4,7 Angiostatin, a 38-kDa internal fragment of plasminogen, is an endogenous inhibitor of angiogenesis.10 This protein has been found in the serum and urine of mice with Lewis lung carcinoma and has been shown to potently inhibit endothelial cell proliferation in vitro and tumor growth in vivo.10 –12 However, given its antitumor mechanism, it is thought that a large quantity of angiostatin would be necessary for cancer therapy. If patients receive this therapy for several years, a shortage of recombinant angiostatin will eventually develop. Gene therapy would seem to solve this problem. Recently, many researchers have investigated the potential of transduction of cDNA encoding angiostatin as a novel therapeutic strategy for several kinds of tumors, including fibrosarcoma, breast cancer, Kaposi’s sarcoma, melanoma, and glioma.16 –21 To our knowledge, the 976

present study is the first investigation of angiostatin gene therapy as a treatment for renal cancer. To evaluate the potential of angiostatin gene therapy for renal cancer, we chose a murine renal cancer cell line, Renca, derived from a murine renal carcinoma that spontaneously arose in a BALB/c mouse.22 In the present study, very rapid regression in remote secondary implanted tumors exposed to angiostatin was observed between 17 and 21 days (Fig. 2B). These tumors seemed to be necrotic macroscopically. This response was observed on the day when maximal tumor growth was noted in the control cells. There are two explanations for this dramatic and delayed response. First, it is considered that an effectual quantity of circulating angiostatin could be gained by growth of tumor transfected with angiostatin after day 17. Second, it is suggested that a rapid ischemic change in the tumors was caused by angiostatin after day 17 when the tumors needed rapid neovascularization to grow at maximal speed. This may be one of the reasons why the growth inhibition of remote secondary implanted tumor was more prominent than that of tumor transfected with angiostatin gene on day 21. However, these tumors could not be completely rejected, and all the mice finally died of tumor growth. A possible explanation for these results is that upregulation of angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor,5,23,24 UROLOGY 59 (6), 2002

would oppose the inhibitory effect of angiostatin. Combination therapy, including inactivation of these angiogenic factors, is a possible solution to this problem. Another possible explanation is that the expression level of angiostatin may not be enough to completely arrest tumor growth. In many studies, the effectiveness of antiangiogenic gene therapy has been limited because stable and adequate transgene expression could not be achieved and maintained for a long duration.16,20,21 In gene therapy for cancer, proteins that can supply antitumor effects by these systematic deliveries, such as angiostatin and endostatin,25 is not always required to be expressed in the tumors. Systemic adenovirus or liposome-mediated administration25–27 or intramuscular administration of expression plasmid vector28 may be used for angiostatin gene therapy in clinical situations. CONCLUSIONS Expression of an angiostatin transgene in vivo can suppress the growth of a murine renal cancer by inhibiting tumor-induced angiogenesis. The delivery of angiostatin by gene therapy may be effective against advanced renal cancer. REFERENCES 1. Motzer RJ, Bander NH, and Nanus DM: Renal-cell carcinoma. N Engl J Med 335: 865– 875, 1996. 2. Motzer RJ, and Russo P: Systemic therapy for renal cell carcinoma. J Urol 163: 408 – 417, 2000. 3. Tsui KH, Shvarts O, Smith RB, et al: Renal cell carcinoma: prognostic significance of incidentally detected tumors. J Urol 163: 426 – 430, 2000. 4. Sabo E, Boltenko A, Sova Y, et al: Microscopic analysis and significance of vascular architectural complexity in renal cell carcinoma. Clin Cancer Res 7: 533–537, 2001. 5. Nicol D, Hii SI, Walsh M, et al: Vascular endothelial growth factor expression is increased in renal cell carcinoma. J Urol 157: 1482–1486, 1997. 6. Tomisawa M, Tokunaga T, Oshika Y, et al: Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma. Eur J Cancer 35: 133–137, 1999. 7. Nativ O, Sabo E, Reiss A, et al: Clinical significance of tumor angiogenesis in patients with localized renal cell carcinoma. Urology 51: 693– 696, 1998. 8. Thelen P, Hemmerlein B, Kugler A, et al: Quantification by competitive quantitative RT-PCR of VEGF121 and VEGF165 in renal cell carcinoma. Anticancer Res 19: 1563– 1565, 1999. 9. Beecken WD, Fernandez A, Joussen AM, et al: Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice. J Natl Cancer Inst 93: 382–387, 2001. 10. O’Reilly MS, Holmgren L, Shing Y, et al: Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79: 315–328, 1994. 11. Sim BK, O’Reilly MS, Liang H, et al: A recombinant

UROLOGY 59 (6), 2002

human angiostatin protein inhibits experimental primary and metastatic cancer. Cancer Res 57: 1329 –1334, 1997. 12. Wu Z, O’Reilly MS, Folkman J, et al: Suppression of tumor growth with recombinant murine angiostatin. Biochem Biophys Res Commun 236: 651– 654, 1997. 13. Dong Z, Yoneda J, Kumar R, et al: Angiostatin-mediated suppression of cancer metastases by primary neoplasms engineered to produce granulocyte/macrophage colony-stimulating factor. J Exp Med 188: 755–763, 1998. 14. Griscelli F, Li H, Bennaceur-Griscelli A, et al: Angiostatin gene transfer: inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest. Proc Natl Acad Sci USA 95: 6367– 6372, 1998. 15. Claesson-Welsh L, Welsh M, Ito N, et al: Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc Natl Acad Sci USA 95: 5579 –5583, 1998. 16. Cao Y, O’Reilly MS, Marshall B, et al: Expression of angiostatin cDNA in a murine fibrosarcoma suppresses primary tumor growth and produces long-term dormancy of metastases. J Clin Invest 101: 1055–1063, 1998. 17. Chen QR, Kumar D, Stass SA, et al: Liposomes complexed to plasmids encoding angiostatin and endostatin inhibit breast cancer in nude mice. Cancer Res 59: 3308 –3312, 1999. 18. Indraccolo S, Morini M, Gola E, et al: Effects of angiostatin gene transfer on functional properties and in vivo growth of Kaposi’s sarcoma cells. Cancer Res 61: 5441–5446, 2001. 19. Sacco MG, Caniatti M, Cato EM, et al: Liposome-delivered angiostatin strongly inhibits tumor growth and metastasization in a transgenic model of spontaneous breast cancer. Cancer Res 60: 2660 –2665, 2000. 20. Tanaka T, Cao Y, Folkman J, et al: Viral vector-targeted antiangiogenic gene therapy utilizing an angiostatin complementary DNA. Cancer Res 58: 3362–3369, 1998. 21. Ambs S, Dennis S, Fairman J, et al: Inhibition of tumor growth correlates with the expression level of a human angiostatin transgene in transfected B16F10 melanoma cells. Cancer Res 59: 5773–5777, 1999. 22. Murphy GP, and Hrushesky WJ: A murine renal cell carcinoma. J Natl Cancer Inst 50: 1013–1025, 1973. 23. Bussolino F, Di Renzo MF, Ziche M, et al: Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 119: 629 – 641, 1992. 24. Yoshimura K, Eto H, Miyake H, et al: Messenger ribonucleic acids for fibroblast growth factors and their receptor in bladder and renal cell carcinoma cell lines. Cancer Lett 103: 91–97, 1996. 25. Sauter BV, Martinet O, Zhang WJ, et al: Adenovirusmediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases. Proc Natl Acad Sci USA 97: 4802– 4807, 2000. 26. Rodolfo M, Cato EM, Soldati S, et al: Growth of human melanoma xenografts is suppressed by systemic angiostatin gene therapy. Cancer Gene Ther 8: 491– 496, 2001. 27. Wen XY, Bai Y, and Stewart AK: Adenovirus-mediated human endostatin gene delivery demonstrates strain-specific antitumor activity and acute dose-dependent toxicity in mice. Hum Gene Ther 12: 347–358, 2001. 28. Blezinger P, Wang J, Gondo M, et al: Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. Nat Biotechnol 17: 343–348, 1999.

977