The effect of alpha-v integrin inhibition on the malignant characteristics of medulloblastoma

Laboratory investigation

Restricted access

Object

Hypoxia induces an aggressive phenotype in some brain tumors in part due to hypoxia-inducible factor–1α (HIF-1α) and integrin expression. The importance of hypoxia in medulloblastoma is unclear and the interaction of HIF-1α and c-Myc in medulloblastoma has not been explored. The objective of this study was to determine if hypoxia induces an aggressive phenotype in human medulloblastoma cells that constitutively express high (D283 Med) or low (DAOY) levels of c-Myc and to determine if blocking αv integrins with the monoclonal antibody intetumumab inhibits hypoxia-induced cellular stress responses.

Methods

Cells were grown at 21% and 1% O2 and in the presence or absence of intetumumab. Measures of malignancy evaluated included cell proliferation, cell migration, and expression of vascular endothelial growth factor (VEGF), αv integrins, HIF-1α, and c-Myc.

Results

Both cell lines robustly expressed αv integrins. Hypoxic DAOY cells showed significantly increased proliferation compared with normoxic controls (p < 0.05), whereas D283 Med cells did not. Both cell lines exhibited a dose-dependent decrease in proliferation when treated with intetumumab (p < 0.05). Hypoxia did not increase DAOY migration, but intetumumab significantly inhibited migration at both oxygen conditions (p < 0.05). Intetumumab significantly decreased VEGF levels in DAOY cells at both oxygen conditions (p < 0.05) and in normoxic D283 cells (p < 0.01). Neither cell line demonstrated increased HIF-1α expression in response to hypoxia. However, hypoxic D283 Med cells grown in the presence of intetumumab demonstrated significantly decreased c-Myc expression (p < 0.05).

Conclusions

Hypoxia did not clearly induce a more aggressive phenotype in medulloblastoma cells. Despite this result, intetumumab decreased medulloblastoma cell proliferation and migration and variably decreased VEGF and c-Myc expression in hypoxic conditions. Targeting αv integrins represents a promising potential adjuvant modality in the treatment of medulloblastoma, particularly subtypes that metastasize and overexpress VEGF and c-Myc.

Abbreviations used in this paper:DMEM = Dulbecco modification of Eagle medium; ELISA = enzyme-linked immunosorbent assay; FBS = fetal bovine serum; HIF = hypoxia-inducible factor; PBS = phosphate-buffered saline; VEGF = vascular endothelial growth factor.

Article Information

Address correspondence to: Edward A. Neuwelt, M.D., Department of Neurology, Blood-Brain Barrier Program, Oregon Health & Science University, 3181 Sam Jackson Park Road, L603, Portland, Oregon 97239. email: neuwelte@ohsu.edu.

Please include this information when citing this paper: published online October 19, 2012; DOI: 10.3171/2012.9.PEDS12268.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    DAOY and D283 Med cells express αv integrins. A: Graph showing DAOY cells express αv, αvβ3 and αvβ5 integrins on the cell surface in vitro. B: Graph showing D283 Med cells express αv, αvβ5 and αvβ1 integrins on the cell surface in vitro. In A and B, data represent the mean of 2 different experiments, and values are adjusted to the background level of fluorescence. Error bars = standard error. C: Representative Western blot demonstrates robust expression of αv integrins in both DAOY and D283 Med cell lines. There was no significant difference in the amount of αv integrin expression between the 2 cell lines, between cells grown at 1% or 21% O2, or cells treated with intetumumab (Int, +) or vehicle (−). RFU = relative fluorescent unit.

  • View in gallery

    Proliferative response to hypoxia differs in DAOY and D283 Med cells and is inhibited by intetumumab. Left: Graph representing DAOY cells grown in 1% (white bars) or 21% O2 (gray bars) in the presence of 3 escalating doses of intetumumab. Cells grown in the presence of 1.5 mg/ml intetumumab showed significantly less proliferation compared with cells grown in the presence of vehicle in their respective oxygen condition. Cells grown at 1% O2 demonstrated significantly more proliferation compared with their counterparts grown at 21% O2 with the exception of cells grown in the presence of 1.5 mg/ml intetumumab. Right: Graph representing D283 Med cells grown under 1% or 21% O2 conditions at 3 escalating doses of intetumumab. Cells grown in the presence of all doses of intetumumab showed significantly less proliferation compared with cells grown in the presence of vehicle within their respective oxygen condition. There was no difference in proliferation between cells grown at 1% and 21% O2. Figures are means of 3 different experiments. Error bars = standard error. *p < 0.05, **p < 0.01; NS = not significant.

  • View in gallery

    c-Myc expression in D283 Med cells is inhibited by the combination of hypoxia and intetumumab. A: Representative Western blot demonstrates decreased expression of c-Myc in DAOY cells compared with D283 Med cells and D283 Med cells grown at 1% O2 in the presence of intetumumab (+). B: Graph of the expression of c-Myc in 3 separate experiments normalized to tubulin, in which the expression appears greater in D283 Med cells than in DAOY cells, although this difference was not significant due to large variance. C: Graph of the expression of c-Myc in 3 separate experiments, normalized to tubulin and to the control (21% O2 vehicle). Neither hypoxic conditions nor the presence of intetumumab significantly affected the expression of c-Myc in DAOY cells. D283 Med cells grown in 1% O2 in the presence of intetumumab demonstrated significantly less c-Myc expression compared with controls. Error bars = standard error. White bars = 1% O2, gray bars = 21% O2. *p < 0.05; AU = arbitrary units.

  • View in gallery

    Intetumumab inhibits VEGF but not HIF-1α expression in DAOY and D283 Med cells. A: Graph showing intetumumab significantly decreased VEGF production in DAOY cells grown at both oxygen conditions and D283 Med cells grown at 21% O2 in 2 separate experiments. B: Graph showing there was no significant difference in the production of HIF-1α in cells grown in the presence of intetumumab or hypoxic conditions in both DAOY and D283 Med cell lines. C: Representative Western blot demonstrates no significant difference in HIF-1α expression in both cell lines grown in various conditions. Error bars = standard error. Gray bars = 21% O2, white bars = 1% O2. *p < 0.05.

  • View in gallery

    Cell migration is inhibited by intetumumab. Upper: DAOY cell migration in both hypoxic and normoxic conditions was significantly inhibited by intetumumab. There was no significant difference in rate of migration between the two oxygen environments. Lower: Representative photos of intetumumab inhibiting cell migration (gap closure) at 0-, 8-, and 24-hour time points. Original magnification ×50. *p < 0.05.

References

  • 1

    Brandsma DUlfman LReijneveld JCBracke MTaphoorn MJZwaginga JJ: Constitutive integrin activation on tumor cells contributes to progression of leptomeningeal metastases. Neuro Oncol 8:1271362006

    • Search Google Scholar
    • Export Citation
  • 2

    Brat DJCastellano-Sanchez AAHunter SBPecot MCohen CHammond EH: Pseudopalisades in glioblastoma are hypoxic, express extracellular matrix proteases, and are formed by an actively migrating cell population. Cancer Res 64:9209272004

    • Search Google Scholar
    • Export Citation
  • 3

    Chu FMPicus JFracasso PMDreicer RLang ZFoster B: A phase 1, multicenter, open-label study of the safety of two dose levels of a human monoclonal antibody to human α(v) integrins, intetumumab, in combination with docetaxel and prednisone in patients with castrate-resistant metastatic prostate cancer. Invest New Drugs 29:6746792011

    • Search Google Scholar
    • Export Citation
  • 4

    Fiorilli PPartridge DStaniszewska IWang JYGrabacka MSo K: Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation. Lab Invest 88:114311562008

    • Search Google Scholar
    • Export Citation
  • 5

    Friedlander MBrooks PCShaffer RWKincaid CMVarner JACheresh DA: Definition of two angiogenic pathways by distinct alpha v integrins. Science 270:150015021995

    • Search Google Scholar
    • Export Citation
  • 6

    Friedman HSBurger PCBigner SHTrojanowski JQWikstrand CJHalperin EC: Establishment and characterization of the human medulloblastoma cell line and transplantable xenograft D283 Med. J Neuropathol Exp Neurol 44:5926051985

    • Search Google Scholar
    • Export Citation
  • 7

    Gordan JDThompson CBSimon MC: HIF and c-Myc: sibling rivals for control of cancer cell metabolism and proliferation. Cancer Cell 12:1081132007

    • Search Google Scholar
    • Export Citation
  • 8

    Gupta RChetty CBhoopathi PLakka SMohanam SRao JS: Downregulation of uPA/uPAR inhibits intermittent hypoxia-induced epithelial-mesenchymal transition (EMT) in DAOY and D283 medulloblastoma cells. Int J Oncol 38:7337442011

    • Search Google Scholar
    • Export Citation
  • 9

    Harris AL: Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer 2:38472002

  • 10

    Huang LE: Carrot and stick: HIF-alpha engages c-Myc in hypoxic adaptation. Cell Death Differ 15:6726772008

  • 11

    Hynes RO: Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11251992

  • 12

    Jennings MTCmelak AJohnson MDMoots PLPais RShyr Y: Differential responsiveness among “high risk” pediatric brain tumors in a pilot study of dose-intensive induction chemotherapy. Pediatr Blood Cancer 43:46542004

    • Search Google Scholar
    • Export Citation
  • 13

    Koshiji MKageyama YPete EAHorikawa IBarrett JCHuang LE: HIF-1alpha induces cell cycle arrest by functionally counteracting Myc. EMBO J 23:194919562004

    • Search Google Scholar
    • Export Citation
  • 14

    Leary SEOlson JM: The molecular classification of medulloblastoma: driving the next generation clinical trials. Curr Opin Pediatr 24:33392012

    • Search Google Scholar
    • Export Citation
  • 15

    Li LWelser JVMilner R: Absence of the alpha v beta 3 integrin dictates the time-course of angiogenesis in the hypoxic central nervous system: accelerated endothelial proliferation correlates with compensatory increases in alpha 5 beta 1 integrin expression. J Cereb Blood Flow Metab 30:103110432010

    • Search Google Scholar
    • Export Citation
  • 16

    Li QKluz TSun HCosta M: Mechanisms of c-myc degradation by nickel compounds and hypoxia. PLoS ONE 4:e85312009

  • 17

    Lim MGuccione SHaddix TSims LCheshier SChu P: alpha(v)beta(3) Integrin in central nervous system tumors. Hum Pathol 36:6656692005

    • Search Google Scholar
    • Export Citation
  • 18

    Lu HLi YShu MTang JHuang YZhou Y: Hypoxiainducible factor-1alpha blocks differentiation of malignant gliomas. FEBS J 276:729173042009

    • Search Google Scholar
    • Export Citation
  • 19

    MacDonald TJBrown KMLaFleur BPeterson KLawlor CChen Y: Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease. Nat Genet 29:1431522001

    • Search Google Scholar
    • Export Citation
  • 20

    MacDonald TJStewart CFKocak MGoldman SEllenbogen RGPhillips P: Phase I clinical trial of cilengitide in children with refractory brain tumors: Pediatric Brain Tumor Consortium Study PBTC-012. J Clin Oncol 26:9199242008

    • Search Google Scholar
    • Export Citation
  • 21

    Mullamitha SATon NCParker GJJackson AJulyan PJRoberts C: Phase I evaluation of a fully human antialphav integrin monoclonal antibody (CNTO 95) in patients with advanced solid tumors. Clin Cancer Res 13:212821352007

    • Search Google Scholar
    • Export Citation
  • 22

    Nisato RETille JCJonczyk AGoodman SLPepper MS: alphav beta 3 and alphav beta 5 integrin antagonists inhibit angiogenesis in vitro. Angiogenesis 6:1051192003

    • Search Google Scholar
    • Export Citation
  • 23

    Northcott PAKorshunov APfister SMTaylor MD: The clinical implications of medulloblastoma subgroups. Nat Rev Neurol 8:3403512012

    • Search Google Scholar
    • Export Citation
  • 24

    Northcott PAKorshunov AWitt HHielscher TEberhart CGMack S: Medulloblastoma comprises four distinct molecular variants. J Clin Oncol 29:140814142011

    • Search Google Scholar
    • Export Citation
  • 25

    O'Day SPavlick ALoquai CLawson DGutzmer RRichards J: A randomised, phase II study of intetumumab, an anti-αv-integrin mAb, alone and with dacarbazine in stage IV melanoma. Br J Cancer 105:3463522011

    • Search Google Scholar
    • Export Citation
  • 26

    Okuyama HEndo HAkashika TKato KInoue M: Downregulation of c-MYC protein levels contributes to cancer cell survival under dual deficiency of oxygen and glucose. Cancer Res 70:10213102232010

    • Search Google Scholar
    • Export Citation
  • 27

    Pereira ERLiao NNeale GAHendershot LM: Transcriptional and post-transcriptional regulation of proangiogenic factors by the unfolded protein response. PLoS ONE 5:e125212010

    • Search Google Scholar
    • Export Citation
  • 28

    Pistollato FRampazzo EPersano LAbbadi SFrasson CDenaro L: Interaction of hypoxia-inducible factor-1α and Notch signaling regulates medulloblastoma precursor proliferation and fate. Stem Cells 28:191819292010

    • Search Google Scholar
    • Export Citation
  • 29

    Rutka JTGiblin JDougherty DVMcCulloch JRDeArmond SJRosenblum ML: An ultrastructural and immunocytochemical analysis of leptomeningeal and meningioma cultures. J Neuropathol Exp Neurol 45:2853031986

    • Search Google Scholar
    • Export Citation
  • 30

    Schwartz DLBankson JBidaut LHe YWilliams RLemos R: HIF-1-dependent stromal adaptation to ischemia mediates in vivo tumor radiation resistance. Mol Cancer Res 9:2592702011

    • Search Google Scholar
    • Export Citation
  • 31

    Siu IMLal ABlankenship JRAldosari NRiggins GJ: c-Myc promoter activation in medulloblastoma. Cancer Res 63:477347762003

  • 32

    Skuli NMonferran SDelmas CFavre GBonnet JToulas C: Alphavbeta3/alphavbeta5 integrins-FAK-RhoB: a novel pathway for hypoxia regulation in glioblastoma. Cancer Res 69:330833162009

    • Search Google Scholar
    • Export Citation
  • 33

    Trikha MZhou ZNemeth JAChen QSharp CEmmell E: CNTO 95, a fully human monoclonal antibody that inhibits alphav integrins, has antitumor and antiangiogenic activity in vivo. Int J Cancer 110:3263352004

    • Search Google Scholar
    • Export Citation
  • 34

    Verma STavaré CJGilles FH: Histologic features and prognosis in pediatric medulloblastoma. Pediatr Dev Pathol 11:3373432008

  • 35

    Wang WMacaulay RJ: Cell-cycle gene expression in lovastatin-induced medulloblastoma apoptosis. Can J Neurol Sci 30:3493572003

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 191 191 97
Full Text Views 80 76 0
PDF Downloads 181 159 0
EPUB Downloads 0 0 0

PubMed

Google Scholar