Cooperative Inhibitory Effect Of Bevacizumab And Nutlin-3a on Tumor Growth, Angiogenesis, And Metastasis In An Experimental Model Of Neuroblastoma

Abstract

Introduction: Vascular endothelial growth factor (VEGF) is critical to tumor angiogenesis, tumor growth and metastasis. However, in neuroblastoma, bevacizumab, a humanized anti-VEGF antibody, has demonstrated limited efficacy alone or in combination with chemotherapy. The oncogene MDM2, a major negative regulator of p53, is also a regulator of hypoxia inducible factor-1α (HIF-1α), and inhibition of MDM2 has been shown in vitro to suppress HIF-1α as well as downstream VEGF expression. We have previously demonstrated that MDM2 inhibition with the small molecule inhibitor, Nutlin-3a, suppresses tumor angiogenesis and tumor growth in an in vivo, orthotopic model of neuroblastoma. Therefore we hypothesize that bevacizumab in combination with Nutlin-3a leads to cooperative inhibition of tumor angiogenesis, tumor growth, and metastasis in an in vivo model of neuroblastoma. Methods: Forty nude mice were intrarenally implanted with the human neuroblastoma cell line, SH-SY5Y. Two weeks after tumor implantation, mice were administered respective treatments for two weeks; control (Nutlin vehicle and bevacizumab vehicle), Nutlin-3a (200 mg/kg/dose BID x 14 days), bevacizumab (5mg/kg/dose BIW x 2 weeks), combination (Nutlin-3a and bevacizumab as above). After two weeks of treatment, all mice were sacrificed. At necropsy, tumors were resected, weighed and preserved. Livers from all mice were resected and preserved. Angiogenesis of preserved xenograft tumors was evaluated by immunohistochemistry for vascular markers, such as endothelial cells (CD-31), perivascular basement membrane (type-IV collagen), and vascular mural cells (αSMA). Tumors were also evaluated for apoptosis. All immunostained tumor tissue underwent digital quantification. Preserved liver tissue underwent H&E staining and histologic analysis for metastasis. Results: The combination of Nutlin-3a and bevacizumab more effectively inhibited xenograft tumor growth than either agent alone when compared to control treatment (79% suppression, p=0.01). Digital quantification of immunostained tumor tissue treated with combination therapy demonstrated significant inhibition of endothelial cells, perivascular basement membrane, and vascular mural cells compared to control treatment tumors (Figure 1). Nutlin-3a alone and in combination with bevacizumab led to significant tumor apoptosis and a significant decrease in both the incidence of metastasis and metastatic burden. Conclusions: We conclude that bevacizumab combined with Nutlin-3a cooperatively inhibits tumor growth, tumor angiogenesis, and metastasis in neuroblastoma. By targeting VEGF and p53 pathways, this novel anti-angiogenic regimen may provide more effective tumor suppression in patients with neuroblastoma and other p53 wild-type tumors.