title: STAT-1 expression is regulated by IGFBP-3 in malignant glioma cells and is a strong predictor of poor survival in patients with glioblastoma.
abstract: OBJECTIVE
  Insulin-like growth factor binding proteins (IGFBPs) have been implicated in the pathogenesis of glioma. In a previous study the authors demonstrated that IGFBP-3 is a novel glioblastoma biomarker associated with poor survival. Since signal transducer and activator of transcription 1 (STAT-1) has been shown to be regulated by IGFBP-3 during chondrogenesis and is a prosurvival and radioresistant molecule in different tumors, the aim in the present study was to explore the functional significance of IGFBP-3 in malignant glioma cells, to determine if STAT-1 is indeed regulated by IGFBP-3, and to study the potential of STAT-1 as a biomarker in glioblastoma.
  
  
  METHODS
  The functional significance of IGFBP-3 was investigated using the short hairpin (sh)RNA gene knockdown approach on U251MG cells. STAT-1 regulation by IGFBP-3 was tested on U251MG and U87MG cells by shRNA gene knockdown and exogenous treatment with recombinant IGFBP-3 protein. Subsequently, the expression of STAT-1 was analyzed with real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) in glioblastoma and control brain tissues. Survival analyses were done on a uniformly treated prospective cohort of adults with newly diagnosed glioblastoma (136 patients) using Kaplan-Meier and Cox regression models.
  
  
  RESULTS
  IGFBP-3 knockdown significantly impaired proliferation, motility, migration, and invasive capacity of U251MG cells in vitro (p < 0.005). Exogenous overexpression of IGFBP-3 in U251MG and U87MG cells demonstrated STAT-1 regulation. The mean transcript levels (by real-time RT-PCR) and the mean labeling index of STAT-1 (by IHC) were significantly higher in glioblastoma than in control brain tissues (p = 0.0239 and p < 0.001, respectively). Multivariate survival analysis revealed that STAT-1 protein expression (HR 1.015, p = 0.033, 95% CI 1.001-1.029) along with patient age (HR 1.025, p = 0.005, 95% CI 1.008-1.042) were significant predictors of shorter survival in patients with glioblastoma.
  
  
  CONCLUSIONS
  IGFBP-3 influences tumor cell proliferation, migration, and invasion and regulates STAT-1 expression in malignant glioma cells. STAT-1 is overexpressed in human glioblastoma tissues and emerges as a novel prognostic biomarker.
authors.affiliation: Departments of Neuropathology.
authors.forename: Balaram
authors.initials: B
authors.lastname: Thota
authors.forename: Arivazhagan
authors.initials: A
authors.lastname: Arimappamagan
authors.forename: Thennarasu
authors.initials: T
authors.lastname: Kandavel
authors.forename: Arun H
authors.initials: AH
authors.lastname: Shastry
authors.forename: Paritosh
authors.initials: P
authors.lastname: Pandey
authors.forename: Bangalore Ashwathnarayanarao
authors.initials: BA
authors.lastname: Chandramouli
authors.forename: Alangar Sathyaranjandas
authors.initials: AS
authors.lastname: Hegde
authors.forename: Paturu
authors.initials: P
authors.lastname: Kondaiah
authors.forename: Vani
authors.initials: V
authors.lastname: Santosh
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics; D001932:Brain Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000401:mortality; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D005260:Female; D005909:Glioblastoma / Q000235:genetics* / Q000401:mortality; D005910:Glioma / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics* / Q000502:physiology; D008297:Male; D008875:Middle Aged; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology; D011237:Predictive Value of Tests; D050794:STAT1 Transcription Factor / Q000096:biosynthesis / Q000235:genetics*; D016019:Survival Analysis
chemical_list: D014408:Biomarkers, Tumor; D018972:Insulin-Like Growth Factor Binding Protein 3; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human
keywords: DMEM = Dulbecco's modified Eagle's medium; FBS = fetal bovine serum; GBM = glioblastoma; HRP = horseradish peroxidase; IFN = interferon; IGF = insulin-like growth factor; IGFBP = IGF binding protein; IGFBP-3; IHC = immunohistochemistry; IR = insulin receptor; KPS = Karnofsky Performance Scale; LI = labeling index; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD = optical density; STAT-1; STAT-1 = signal transducer and activator of transcription 1; TGFβ = transforming growth factor–β; biomarker; glioblastoma; oncology; prognosis; qPCR = quantitative polymerase chain reaction; shRNA = short hairpin RNA