title: Effect of MDM2 and vascular endothelial growth factor inhibition on tumor angiogenesis and metastasis in neuroblastoma.
abstract: Neuroblastoma is the most common pediatric abdominal tumor and principally a p53 wild-type, highly vascular, aggressive tumor, with limited response to anti-VEGF therapies alone. MDM2 is a key inhibitor of p53 and a positive activator of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) activity with an important role in neuroblastoma pathogenesis. We hypothesized that concurrent inhibition of both MDM2 and VEGF signaling would have cooperative anti-tumor effects, potentiating anti-angiogenic strategies for neuroblastoma and other p53 wild-type tumors. We orthotopically implanted SH-SY5Y neuroblastoma cells into nude mice (n = 40) and treated as follows: control, bevacizumab, Nutlin-3a, combination of bevacizumab plus Nutlin-3a. Expression of HIF-1α and VEGF were measured by qPCR, Western blot, and ELISA. Tumor apoptosis was measured by immunohistochemistry and caspase assay. Angiogenesis was evaluated by immunohistochemistry for vascular markers (CD-31, type-IV collagen, αSMA). Both angiogenesis and metastatic burden were digitally quantified. In vitro, Nutlin-3a suppresses HIF-1α expression with subsequent downregulation of VEGF. Bevacizumab plus Nutlin-3a leads to significant suppression of tumor growth compared to control (P < 0.01) or either agent alone. Combination treated xenograft tumors display a marked decrease in endothelial cells (P < 0.0001), perivascular basement membrane (P < 0.04), and vascular mural cells (P < 0.004). Nutlin-3a alone and in combination with bevacizumab leads to significant tumor apoptosis (P < 0.0001 for both) and significant decrease in incidence of metastasis (P < 0.05) and metastatic burden (P < 0.03). Bevacizumab plus Nutlin-3a cooperatively inhibits tumor growth and angiogenesis in neuroblastoma in vivo with dramatic effects on tumor vascularity. Concomitantly targeting VEGF and p53 pathways potently suppresses tumor growth, and these results support further clinical development of this approach.
authors.affiliation: Michael E. Debakey Department of Surgery, Division of Pediatric Surgery, Baylor College of Medicine, Texas Children's Hospital, Houston, 77030, USA.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Patterson
authors.forename: Dongbing
authors.initials: D
authors.lastname: Gao
authors.forename: Denae N
authors.initials: DN
authors.lastname: Trahan
authors.forename: Brett A
authors.initials: BA
authors.lastname: Johnson
authors.forename: Andrew
authors.initials: A
authors.lastname: Ludwig
authors.forename: Eveline
authors.initials: E
authors.lastname: Barbieri
authors.forename: Zaowen
authors.initials: Z
authors.lastname: Chen
authors.forename: Jose
authors.initials: J
authors.lastname: Diaz-Miron
authors.forename: Lyubomir
authors.initials: L
authors.lastname: Vassilev
authors.forename: Jason M
authors.initials: JM
authors.lastname: Shohet
authors.forename: Eugene S
authors.initials: ES
authors.lastname: Kim
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D000068258:Bevacizumab; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics; D007093:Imidazoles / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D009362:Neoplasm Metastasis; D009389:Neovascularization, Pathologic / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009447:Neuroblastoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D020533:Angiogenesis Inhibitors; D061067:Antibodies, Monoclonal, Humanized; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007093:Imidazoles; D010879:Piperazines; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2