title: Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion.
abstract: BACKGROUND
  Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets.
  
  
  METHODS
  Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin β1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided.
  
  
  RESULTS
  Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-κB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF(200 ng/mL): 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-κB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF(200 ng/mL) + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF.
  
  
  CONCLUSIONS
  A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.
authors.affiliation: Neuro-Oncology Branch, National Cancer Institute/National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-1002, USA.
authors.forename: Lincoln A
authors.initials: LA
authors.lastname: Edwards
authors.forename: Kevin
authors.initials: K
authors.lastname: Woolard
authors.forename: Myung Jin
authors.initials: MJ
authors.lastname: Son
authors.forename: Aiguo
authors.initials: A
authors.lastname: Li
authors.forename: Jeongwu
authors.initials: J
authors.lastname: Lee
authors.forename: Chibawanye
authors.initials: C
authors.lastname: Ene
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Mantey
authors.forename: Dragan
authors.initials: D
authors.lastname: Maric
authors.forename: Hua
authors.initials: H
authors.lastname: Song
authors.forename: Galina
authors.initials: G
authors.lastname: Belova
authors.forename: Robert T
authors.initials: RT
authors.lastname: Jensen
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Howard A
authors.initials: HA
authors.lastname: Fine
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology*; D015820:Cadherins / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement; D047369:Chromatin Immunoprecipitation; D055513:Connective Tissue Growth Factor / Q000378:metabolism*; D004195:Disease Models, Animal; D004789:Enzyme Activation; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique; D005910:Glioma / Q000378:metabolism* / Q000473:pathology*; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D019012:Integrin beta1 / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D020932:Nerve Growth Factor / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020917:Receptor, trkA / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism*; D014183:Transplantation, Heterologous; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: D015820:Cadherins; D018398:Homeodomain Proteins; D019012:Integrin beta1; D007457:Iodine Radioisotopes; D016328:NF-kappa B; D034741:RNA, Small Interfering; D014157:Transcription Factors; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1; D055513:Connective Tissue Growth Factor; D020932:Nerve Growth Factor; D008156:Luciferases; D020917:Receptor, trkA