Cancer Therapy: Preclinical Sunitinib Inhibits Tumor Growth and Synergizes with Cisplatin in Orthotopic Models of Cisplatin-Sensitive and Cisplatin-Resistant Human Testicular Germ Cell Tumors

Purpose: Germ cell tumors (GCT) of the testis are highly curable, but those patients who are refractory to cisplatin (CDDP)-based combination chemotherapy have a poor prognosis. Therefore, identifying new alternatives for treatment remains a priority. Several studies support an important role for angiogenesis in GCTs, suggesting that antiangiogenic treatment might be a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor inhibitor with antiangiogenic and antitumor activities. In the present study, we evaluated the effect of sunitinib, CDDP, or the combination of both drugs using an orthotopic model of human testicular GCT. Experimental Design: Mice were implanted with four different testicular tumors: a yolk sac, two choriocarcinomas, and a CDDP-resistant choriocarcinoma variant induced in mice by continuous exposure to CDDP. Mice were treated with vehicle, CDDP, sunitinib, or the combination of both drugs and their effects on tumors were analyzed. Results: We observed a significant inhibition in tumor growth accompanied by longer survival after sunitinib treatment. Combination therapy with CDDP significantly enhanced these effects. Sunitinib induced apoptosis, reduced tumor cell proliferation and tumor vasculature, and inhibited vascular endothelial growth factor receptor 1, 2, and 3 and platelet-derived growth factor receptor α phosphorylation without affecting phosphorylation of other tyrosine kinase receptors. More importantly, tumor growth inhibition induced by sunitinib was also observed in the induced CDDP-resistant choriocarcinoma model. Conclusions: Taken together, these results suggest that sunitinib might be a new alternative for treatment of CDDP-refractory patients. Germ cell tumors (GCT) of the testis are the most common solid tumors in men ages 15 to 35 years and represent 95% of tumors arising in the testes (1). Testicular GCTs can be classified as seminoma or nonseminoma according to their histologic characteristics (1). Each group represents ∼50% of GCTs. Nonseminoma tumors include embryonal cell carcinoma, yolk sac tumor, choriocarcinoma, and teratoma (1). Most nonseminoma tumors are composed of two or more of these cell types; seminoma may also be a component (1). GCTs are highly curable even in patients with metastatic disease. Approximately 70% to 80% of patients with metastatic germ cell cancer can be cured after cisplatin (cis-dichlorodiammine platinum or CDDP)-based combination chemotherapy, such as bleomycin, etoposide, and CDDP or etoposide, ifosfamide, and CDDP (2); however, resistance to CDDP treatment may arise. Indeed, two different types of resistance are observed: CDDP-refractory disease, which consists in disease stabilization during treatment followed by disease progression within 4 weeks after CDDP-based chemotherapy, and absolute CDDP-refractory disease in which there is disease progression even during treatment (3). Patients refractory to CDDP-based Authors' Affiliations: Laboratori de Recerca Translacional and Servei d'Oncologia Mèdica, Institut Català d'Oncologia-IDIBELL, Hospital Duran i Reynals, L'Hospitalet de Llobregat; Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge-IDIBELL; Departament de Bioquímica, Hospital de Sant Pau; Departament de Ciències Fisiològiques II, Universitat de Barcelona-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain Received 8/19/08; revised 2/3/09; accepted 2/16/09; published OnlineFirst 5/5/09. Grant support: Marató de TV3 predoctoral fellowship (W. Castillo-Ávila); Spanish Association Against Cancer fellowship (J.M. Piulats); Ministerio de Educación y Ciencia, Spain, Programa Ramón y Cajal, RTICC RD2006-0092, SAF2004-01350, and SAF2007-60955 (F. Viñals) and SAF2002-02265 (A. Villanueva); Ministerio de Sanidad y Consumo, Spain grant FIS2003PI030264 (X. Garcia del Muro); Generalitat de Catalunya, Departament d'Universitat, Recerca i Societat de la Informació grant 2005SGR00727; and Fundació La Marató de TV3 grant 051430 (F. Viñals and X. Garcia del Muro). The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Francesc Viñals, Laboratori de Recerca Translacional, Institut Català d'Oncologia-IDIBELL, Hospital Duran i Reynals, L'Hospitalet de Llobregat, Gran Via s/n km 2,7, 08907 Barcelona, Spain. Phone: 34932607344; Fax: 34-932607466; E-mail: fvinyals@ico.scs.es. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2170 3384 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Research. on April 15, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from combination chemotherapy have a poor prognosis. The identification of new treatment alternatives for patients with refractory disease remains a priority and novel molecular targets are being explored. Antiangiogenic therapy has the potential to be an effective strategy for human cancer treatment. Angiogenesis, or the formation of new blood vessels from preexisting vasculature, is a complex multistep process that includes endothelial cell proliferation, vessel sprouting, vascular permeability, and the remodeling and maturation of emerging vessels. Angiogenesis is essential to support the growth and metastatic dissemination of most solid tumors (4, 5). A balance between proangiogenic and antiangiogenic factors controls this process. Multiple growth factors, including vascular endothelial growth factors (VEGF), fibroblast growth factors, and platelet-derived growth factors (PDGF), exert an important proangiogenic effect through binding of specific cell surface receptor tyrosine kinases (RTK; ref. 6). In addition to ligand activation, somatic mutations also can activate RTKs. Enhanced activity of many RTKs has been implicated in tumor growth, progression, metastasis, and angiogenesis (7). Sunitinib is an oral multitarget RTK inhibitor with antiangiogenic and antitumor activities (8–10). Sunitinib inhibits RTKs expressed by tumor cells and involved in tumor proliferation and survival, including stem cell factor receptor (c-Kit), Fmslike tyrosine kinase 3, colony-stimulating factor type I receptor, and the glial cell line-derived neurotrophic factor receptor (8, 9). Moreover, sunitinib inhibits RTKs expressed on endothelial and mural cells, such as PDGF receptor (PDGFR; α and β) and VEGF receptor (VEGFR; types 1 and 2), which are involved in angiogenesis (8, 10). The antitumor activity of sunitinib has been shown in preclinical and clinical studies (10–15). Tumor regression has been shown in different xenograft models of colon cancer, breast cancer, non-small cell lung cancer, melanoma, glioblastoma, renal carcinoma, and epidermal cancer (10–12). Clinically, sunitinib antitumor activity has been shown in phase I, II, and III trials in patients with renal cancer, gastrointestinal stromal tumors, breast cancer, neuroendocrine tumors, colorectal cancer, sarcoma, thyroid cancer, melanoma, and non-small cell lung cancer (11–15). In addition, sunitinib received U.S. Food and Drug Administration approval in January 2006 and European Union approval in January 2007 for treatment of advanced renal cell carcinoma and patients with gastrointestinal stromal tumors who are refractory or intolerant to imatinib (16, 17). Several reports have shown that some of the RTKs inhibited by sunitinib and their specific ligands are implicated in the development of human testicular GCTs, such as c-Kit, PDGFRs, or VEGFRs (18–20). In the present study, we evaluated the effect of sunitinib on a newly developed model of human testicular GCTs orthotopically grown in nude mice. We used four different tumors types including yolk sac and choriocarcinoma. Because platinum-based drugs are the standard treatment for GCTs, we evaluated the antitumor and antiangiogenic activity of sunitinib alone or in combination with CDDP. Our results indicate that sunitinib clearly inhibited tumor growth, prolonged mice survival, and reduced tumor vasculature. More interestingly, sunitinib was equally effective in a CDDP-resistant model of testicular GCT, suggesting that this drug might be a new alternative for treatment of CDDP-refractory patients in the above different tumor types. Materials and Methods Chemical compounds. Sunitinib was kindly provided by Pfizer and was dissolved in carboxymethylcellulose solution (carboxymethylcellulose 0.5%, NaCl 1.8%, Tween 80 0.4%, and benzyl alcohol 0.9% in distilled water) and adjusted to pH 6.0. Drug aliquots were prepared once weekly and kept in the dark at 4°C. CDDP was diluted in sterile serum before intraperitoneal injection. All other reagents were from Sigma unless stated otherwise. Orthotopic implantation of testicular tumors. Male nu/nu Swiss mice were purchased from Charles River. Mice were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. All the animal studies were approved by the local committee for animal care. Fresh surgical specimens of GCTs of the testis were obtained after surgical resection from the Hospital Universitari de Bellvitge (L'Hospitalet de Llobregat) and Fundació Puigvert and placed in DMEM (Biowhittaker) supplemented with 10% FCS, 50 units/mL penicillin, and 50 μg/mL streptomycin sulfate. Testicular human GCTs were minced and two pieces of each tumor were orthotopically implanted. Briefly, 5-week-old male weighing 18 to 22 g were anesthetized by isoflurane inhalation. A small midline incision was made and the testes were exteriorized. A piece of tumor was then implanted on each testis using prolene 7-0 surgical sutures. The testes were returned to the abdominal cavity and the incision was closed with wound clips. Different tumors were perpetuated in mice by consecutive passages (at least six) according to the growth rate of each tumor type: when an intra-abdominal mass was palpated testes and tumors were exteriorized and a piece of tumor (2-5 mm) was then implanted on testis of a new animal as described before for the primary tumor. Only nonseminoma tumors (mainly choriocarcinomas, embryonal carcinoma, yolk sac, or mixtures) were perpetuated in mice; all pure seminomas present as primary tumors failed to grow as xenografts. For our studies with sunitinib, we use four models of pure nonseminoma GCTs of the testis: a yolk sac (TGT1), two choriocarcinomas (TGT17 and TGT38), and the CDDP-resistant variant of one of the choriocarcinomas (TGT38R), which was developed by continuous exposure of mice to CDDP and shows acquired resistance to this drug. Quantification of circulating tumor markers. Serum levels of α-fetoprotein (AFP; for yolk sac tumors) and the β-subunit of human chorionic gonadotropin (βHCG; for choriocarcinomas) are used as surrogate Translational Relevance Identifying new alternatives for the treatment of patients with testicular germ cell tumors (GCT) refractory to cisplatin (CDDP) chemotherapy remains a priority. In the present study, we evaluated the effect of sunitinib, an oral multitarget tyrosine kinase receptor inhibitor, in a preclinical model of testicular GCTs. Our results indicate that sunitinib has antitumor activity and that combined therapy with CDDP enhances the effect induced by either agent alone. Remarkably, sunitinib was equally effective in a CDDP-resistant model of testicular GCT. Thus, our results suggest that sunitinib might constitute a new alternative for the treatment of CDDP-refractory patients. Moreover, in clinical trials with drugs as sunitinib, it would be interesting to continue the CDDP treatment even after development of drug resistance has taken place. 3385 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Effect of Sunitinib in Orthotopic Testicular GCT Research. on April 15, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from markers of tumor burden (2, 21, 22). They were measured in nude mice serum using commercially available two-site enzyme chemiluminometric assays automated on the Immulite-2000 analyzer. AFP assay uses beads coated with monoclonal murine anti-AFP and alkaline phosphatase conjugated to polyclonal rabbit anti-AFP (23). βHCG assay uses beads coated with monoclonal murine anti-βHCG and alkaline phosphatase conjugated to polyclonal ovine anti-βHCG (24). Reactions were linear up to 300 KU/L for AFP and up to 5,000 units/L for βHCG. Higher concentrations were diluted with the corresponding provided diluents. Assay sensitivity was 0.2 KU/L and 0.4 units/L for AFP and βHCG, respectively. Reference values of ≤9 KU/L for AFP and ≤5 units/L for βHCG were established based on healthy controls. Treatment schedule. Approximately 10 days after tumor implantation in the case of TGT17, TGT38, and TGT38R choriocarcinomas and 30 days after tumor implantation in the case of TGT1 yolk sac tumor, a palpable intra-abdominal mass was detected. Presence of tumor was posteriorly confirmed by detection high serum levels of the surrogate markers human AFP (for yolk sac tumors) and βHCG (for choriocarcinomas) as described before. In this moment, mice were randomized into four treatment groups (n = 10 mice per group in TGT17 choriocarcinoma, n = 7 mice per group in TGT38 and TGT38R choriocarcinomas, and n = 5 mice per group in TGT1): (a) daily oral administration of sunitinib vehicle solution (carboxymethylcellulose suspension) for 15 days and intraperitoneal administration of three doses of physiologic serum at 5-day interval (control group), (b) intraperitoneal administration of three doses of 2 mg/kg CDDP at 5-day intervals (CDDP group), (c) daily oral administration of 40 mg/kg sunitinib for 15 days (sunitinib group), and (d) intraperitoneal administration of three doses of 2 mg/kg CDDP at 5-day intervals and daily oral administration of 40mg/kg sunitinib for 15 days (CDDP and sunitinib group). The chosen doses of 40 mg/kg sunitinib and 2 mg/kg CDDP were found to be the most effective in mice in previous studies (10). Studies were finished when tumors in vehicle-treated animals were judged to adversely affect their well-being (20-25 days after tumor implantation for mice bearing choriocarcinoma tumors and 65-70 days for mice bearing yolk sac tumors). Mice were sacrificed by cervical dislocation and the effect of the different treatments on tumor response was evaluated by tumor volume: volume = (length) (width / 2). For determination of survival time and establishment of KaplanMeier survival curves, mice bearing TGT17 choriocarcinoma were treated with vehicle, 40 mg/kg sunitinib for 15 days, three doses of 2 mg/kg CDDP at 5-day intervals, and the combination of both drugs (5 mice per group). Animals were sacrificed when tumors were judged to affect