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.
Similar content being viewed by others
References
Berthold F, Boos J, Burdach S, Erttmann R, Henze G, Hermann J, Klingebiel T, Kremens B, Schilling FH, Schrappe M, Simon T, Hero B (2005) Myeloablative megatherapy with autologous stem-cell rescue versus oral maintenance chemotherapy as consolidation treatment in patients with high-risk neuroblastoma: a randomised controlled trial. Lancet Oncol 6(9):649–658. doi:10.1016/S1470-2045(05)70291-6
Matthay KK, Villablanca JG, Seeger RC, Stram DO, Harris RE, Ramsay NK, Swift P, Shimada H, Black CT, Brodeur GM, Gerbing RB, Reynolds CP (1999) Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group. N Engl J Med 341(16):1165–1173
Gurney JG, Tersak JM, Ness KK, Landier W, Matthay KK, Schmidt ML (2007) Hearing loss, quality of life, and academic problems in long-term neuroblastoma survivors: a report from the children’s oncology group. Pediatrics 120(5):e1229–e1236. doi:10.1542/peds.2007-0178
Kushner BH, Kramer K, Modak S, Qin LX, Yataghena K, Jhanwar SC, Cheung NK (2009) Reduced risk of secondary leukemia with fewer cycles of dose-intensive induction chemotherapy in patients with neuroblastoma. Pediatr Blood Cancer 53(1):17–22. doi:10.1002/pbc.21931
Laverdiere C, Cheung NK, Kushner BH, Kramer K, Modak S, LaQuaglia MP, Wolden S, Ness KK, Gurney JG, Sklar CA (2005) Long-term complications in survivors of advanced stage neuroblastoma. Pediatr Blood Cancer 45(3):324–332. doi:10.1002/pbc.20331
Matthay KK, Reynolds CP, Seeger RC, Shimada H, Adkins ES, Haas-Kogan D, Gerbing RB, London WB, Villablanca JG (2009) Long-term results for children with high-risk neuroblastoma treated on a randomized trial of myeloablative therapy followed by 13-cis-retinoic acid: a children’s oncology group study. J Clin Oncol 27(7):1007–1013. doi:10.1200/JCO.2007.13.8925
Haupt R, Garaventa A, Gambini C, Parodi S, Cangemi G, Casale F, Viscardi E, Bianchi M, Prete A, Jenkner A, Luksch R, Di Cataldo A, Favre C, D’Angelo P, Zanazzo GA, Arcamone G, Izzi GC, Gigliotti AR, Pastore G, De Bernardi B (1979) Improved survival of children with neuroblastoma between and 2005: a report of the Italian neuroblastoma registry. J Clin Oncol 28(14):2331–2338. doi:10.1200/JCO.2009.24.8351
Chen L, Malcolm AJ, Wood KM, Cole M, Variend S, Cullinane C, Pearson AD, Lunec J, Tweddle DA (2007) p53 is nuclear and functional in both undifferentiated and differentiated neuroblastoma. Cell Cycle 6(21):2685–2696
Vogan K, Bernstein M, Leclerc JM, Brisson L, Brossard J, Brodeur GM, Pelletier J, Gros P (1993) Absence of p53 gene mutations in primary neuroblastomas. Cancer Res 53(21):5269–5273
Freedman DA, Wu L, Levine AJ (1999) Functions of the MDM2 oncoprotein. Cell Mol Life Sci 55(1):96–107
Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid degradation of p53. Nature 387(6630):296–299. doi:10.1038/387296a0
Kubbutat MH, Jones SN, Vousden KH (1997) Regulation of p53 stability by Mdm2. Nature 387(6630):299–303. doi:10.1038/387299a0
Momand J, Jung D, Wilczynski S, Niland J (1998) The MDM2 gene amplification database. Nucleic Acids Res 26(15):3453–3459
Schmidt MK, Tommiska J, Broeks A, van Leeuwen FE, Van’t Veer LJ, Pharoah PD, Easton DF, Shah M, Humphreys M, Dork T, Reincke SA, Fagerholm R, Blomqvist C, Nevanlinna H (2009) Combined effects of single nucleotide polymorphisms TP53 R72P and MDM2 SNP309, and p53 expression on survival of breast cancer patients. Breast Cancer Res 11(6):R89. doi:10.1186/bcr2460
Perfumo C, Parodi S, Mazzocco K, Defferrari R, Inga A, Scarra GB, Ghiorzo P, Haupt R, Tonini GP, Fronza G (2009) MDM2 SNP309 genotype influences survival of metastatic but not of localized neuroblastoma. Pediatr Blood Cancer 53(4):576–583. doi:10.1002/pbc.22132
Willander K, Ungerback J, Karlsson K, Fredrikson M, Soderkvist P, Linderholm M (2010) MDM2 SNP309 promoter polymorphism, an independent prognostic factor in chronic lymphocytic leukemia. Eur J Haematol 85(3):251–256. doi:10.1111/j.1600-0609.2010.01470.x
Chien WP, Wong RH, Cheng YW, Chen CY, Lee H (2010) Associations of MDM2 SNP309, transcriptional activity, mRNA expression, and survival in stage I non-small-cell lung cancer patients with wild-type p53 tumors. Ann Surg Oncol 17(4):1194-1202. doi:10.1245/s10434-009-0853-2
Chen Z, Lin Y, Barbieri E, Burlingame S, Hicks J, Ludwig A, Shohet JM (2009) Mdm2 deficiency suppresses MYCN-Driven neuroblastoma tumorigenesis in vivo. Neoplasia 11(8):753–762
Van Maerken T, Ferdinande L, Taildeman J, Lambertz I, Yigit N, Vercruysse L, Rihani A, Michaelis M, Cinatl J Jr, Cuvelier CA, Marine JC, De Paepe A, Bracke M, Speleman F, Vandesompele J (2009) Antitumor activity of the selective MDM2 antagonist nutlin-3 against chemoresistant neuroblastoma with wild-type p53. J Natl Cancer Inst 101(22):1562–1574. doi:10.1093/jnci/djp355
Kim E, Shohet J (2009) Targeted molecular therapy for neuroblastoma: the ARF/MDM2/p53 axis. J Natl Cancer Inst 101(22):1527–1529. doi:10.1093/jnci/djp376
Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303(5659):844–848. doi:10.1126/science.1092472
Chen F, Wang W, El-Deiry WS (2010) Current strategies to target p53 in cancer. Biochem Pharmacol 80(5):724–730. doi:10.1016/j.bcp.2010.04.031
Brown CJ, Lain S, Verma CS, Fersht AR, Lane DP (2009) Awakening guardian angels: drugging the p53 pathway. Nat Rev Cancer 9(12):862–873. doi:10.1038/nrc2763
Barbieri E, Mehta P, Chen Z, Zhang L, Slack A, Berg S, Shohet JM (2006) MDM2 inhibition sensitizes neuroblastoma to chemotherapy-induced apoptotic cell death. Mol Cancer Ther 5(9):2358–2365. doi:10.1158/1535-7163.MCT-06-0305
Chen D, Li M, Luo J, Gu W (2003) Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function. J Biol Chem 278(16):13595–13598. doi:10.1074/jbc.C200694200
Nieminen AL, Qanungo S, Schneider EA, Jiang BH, Agani FH (2005) Mdm2 and HIF-1alpha interaction in tumor cells during hypoxia. J Cell Physiol 204(2):364–369. doi:10.1002/jcp.20406
Lee YM, Lim JH, Chun YS, Moon HE, Lee MK, Huang LE, Park JW (2009) Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1alpha. Carcinogenesis 30(10):1768–1775. doi:10.1093/carcin/bgp196
Gordan JD, Simon MC (2007) Hypoxia-inducible factors: central regulators of the tumor phenotype. Curr Opin Genet Dev 17(1):71–77. doi:10.1016/j.gde.2006.12.006
Bardos JI, Chau NM, Ashcroft M (2004) Growth factor-mediated induction of HDM2 positively regulates hypoxia-inducible factor 1alpha expression. Mol Cell Biol 24(7):2905–2914
LaRusch GA, Jackson MW, Dunbar JD, Warren RS, Donner DB, Mayo LD (2007) Nutlin3 blocks vascular endothelial growth factor induction by preventing the interaction between hypoxia inducible factor 1alpha and Hdm2. Cancer Res 67(2):450–454. doi:10.1158/0008-5472.CAN-06-2710
Narasimhan M, Rose R, Ramakrishnan R, Zell JA, Rathinavelu A (2008) Identification of HDM2 as a regulator of VEGF expression in cancer cells. Life Sci 82(25–26):1231–1241. doi:10.1016/j.lfs.2008.04.004
Narasimhan M, Rose R, Karthikeyan M, Rathinavelu A (2007) Detection of HDM2 and VEGF co-expression in cancer cell lines: novel effect of HDM2 antisense treatment on VEGF expression. Life Sci 81(17–18):1362–1372. doi:10.1016/j.lfs.2007.08.029
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246(4935):1306–1309
Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, Ferrara N (1993) Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 362(6423):841–844. doi:10.1038/362841a0
Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL, Steinberg SM, Chen HX, Rosenberg SA (2003) A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 349(5):427–434. doi:10.1056/NEJMoa021491
Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350(23):2335–2342. doi:10.1056/NEJMoa032691
Miller KD, Chap LI, Holmes FA, Cobleigh MA, Marcom PK, Fehrenbacher L, Dickler M, Overmoyer BA, Reimann JD, Sing AP, Langmuir V, Rugo HS (2005) Randomized phase III trial of capecitabine compared with bevacizumab plus capecitabine in patients with previously treated metastatic breast cancer. J Clin Oncol 23(4):792–799. doi:10.1200/JCO.2005.05.098
Kreisl TN, Kim L, Moore K, Duic P, Royce C, Stroud I, Garren N, Mackey M, Butman JA, Camphausen K, Park J, Albert PS, Fine HA (2009) Phase II trial of single-agent bevacizumab followed by bevacizumab plus irinotecan at tumor progression in recurrent glioblastoma. J Clin Oncol 27(5):740–745. doi:10.1200/JCO.2008.16.3055
Glade Bender JL, Adamson PC, Reid JM, Xu L, Baruchel S, Shaked Y, Kerbel RS, Cooney-Qualter EM, Stempak D, Chen HX, Nelson MD, Krailo MD, Ingle AM, Blaney SM, Kandel JJ, Yamashiro DJ (2008) Phase I trial and pharmacokinetic study of bevacizumab in pediatric patients with refractory solid tumors: a Children’s Oncology Group Study. J Clin Oncol 26(3):399–405. doi:10.1200/JCO.2007.11.9230
Secchiero P, Corallini F, Gonelli A, Dell’Eva R, Vitale M, Capitani S, Albini A, Zauli G (2007) Antiangiogenic activity of the MDM2 antagonist nutlin-3. Circ Res 100(1):61–69. doi:10.1161/01.RES.0000253975.76198.ff
Slack A, Chen Z, Tonelli R, Pule M, Hunt L, Pession A, Shohet JM (2005) The p53 regulatory gene MDM2 is a direct transcriptional target of MYCN in neuroblastoma. Proc Natl Acad Sci USA 102(3):731–736. doi:10.1073/pnas.0405495102
Wild R, Ramakrishnan S, Sedgewick J, Griffioen AW (2000) Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density. Microvasc Res 59(3):368–376. doi:10.1006/mvre.1999.2233
Kim ES, Serur A, Huang J, Manley CA, McCrudden KW, Frischer JS, Soffer SZ, Ring L, New T, Zabski S, Rudge JS, Holash J, Yancopoulos GD, Kandel JJ, Yamashiro DJ (2002) Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. Proc Natl Acad Sci USA 99(17):11399–11404. doi:10.1073/pnas.172398399
Benesch M, Windelberg M, Sauseng W, Witt V, Fleischhack G, Lackner H, Gadner H, Bode U, Urban C (2008) Compassionate use of bevacizumab (Avastin) in children and young adults with refractory or recurrent solid tumors. Ann Oncol 19(4):807–813. doi:10.1093/annonc/mdm510
Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, Lilenbaum R, Johnson DH (2006) Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355(24):2542–2550. doi:10.1056/NEJMoa061884
Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, Boland P, Leidich R, Hylton D, Burova E, Ioffe E, Huang T, Radziejewski C, Bailey K, Fandl JP, Daly T, Wiegand SJ, Yancopoulos GD, Rudge JS (2002) VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA 99(17):11393–11398. doi:10.1073/pnas.172398299
Mendel DB, Laird AD, Xin X, Louie SG, Christensen JG, Li G, Schreck RE, Abrams TJ, Ngai TJ, Lee LB, Murray LJ, Carver J, Chan E, Moss KG, Haznedar JO, Sukbuntherng J, Blake RA, Sun L, Tang C, Miller T, Shirazian S, McMahon G, Cherrington JM (2003) In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res 9(1):327–337
Lyons JF, Wilhelm S, Hibner B, Bollag G (2001) Discovery of a novel Raf kinase inhibitor. Endocr Relat Cancer 8(3):219–225
Wedge SR, Kendrew J, Hennequin LF, Valentine PJ, Barry ST, Brave SR, Smith NR, James NH, Dukes M, Curwen JO, Chester R, Jackson JA, Boffey SJ, Kilburn LL, Barnett S, Richmond GH, Wadsworth PF, Walker M, Bigley AL, Taylor ST, Cooper L, Beck S, Jurgensmeier JM, Ogilvie DJ (2005) AZD2171: a highly potent, orally bioavailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for the treatment of cancer. Cancer Res 65(10):4389–4400. doi:10.1158/0008-5472.CAN-04-4409
Lorusso P, Shields AF, Gadgeel S, Vaishampayan U, Guthrie T, Puchalski T, Xu J, Liu Q (2010) Cediranib in combination with various anticancer regimens: results of a phase I multi-cohort study. Invest New Drugs. doi:10.1007/s10637-010-9484-5
Loriot Y, Mordant P, Dorvault N, De la Motte Rouge T, Bourhis J, Soria JC, Deutsch E (2010) BMS-690514, a VEGFR and EGFR tyrosine kinase inhibitor, shows anti-tumoural activity on non-small-cell lung cancer xenografts and induces sequence-dependent synergistic effect with radiation. Br J Cancer 103(3):347–353. doi:10.1038/sj.bjc.6605748
Myers AL, Williams RF, Ng CY, Hartwich JE, Davidoff AM (2010) Bevacizumab-induced tumor vessel remodeling in rhabdomyosarcoma xenografts increases the effectiveness of adjuvant ionizing radiation. J Pediatr Surg 45(6):1080–1085. doi:10.1016/jjpedsurg.2010.02.068
Acknowledgments
Funding support: Children’s Neuroblastoma Cancer Foundation (EK); Hankamer Foundation, Baylor College of Medicine (EK); American Cancer Society Research Scholar grant RSG-07-209-01 (JS).
Author information
Authors and Affiliations
Corresponding author
Additional information
J. M. Shohet and E. S. Kim are co-senior authors—both senior authors contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Patterson, D.M., Gao, D., Trahan, D.N. et al. Effect of MDM2 and vascular endothelial growth factor inhibition on tumor angiogenesis and metastasis in neuroblastoma. Angiogenesis 14, 255–266 (2011). https://doi.org/10.1007/s10456-011-9210-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10456-011-9210-8