title: Functional role of CLIC1 ion channel in glioblastoma-derived stem/progenitor cells.
abstract: BACKGROUND
  Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis.
  
  
  METHODS
  We used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane's multiple comparison test. Kaplan-Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided.
  
  
  RESULTS
  CLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1(low) vs CLIC1(high) survival: χ(2) = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient-derived neurospheres.
  
  
  CONCLUSIONS
  Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.
authors.affiliation: Affiliations of authors: Department of Experimental Oncology, European Institute of Oncology, Milan, Italy (MS, DO, CR, PB, LF, GP); Department of Bioscience, University of Milan, Milan, Italy (NS, MA, MM); Department of Anesthesiology, Division of Molecular Medicine, University of California-Los Angeles, Los Angeles, CA (NS); Department of Neurosurgery, Neurocenter, University of Freiburg, Freiburg, Germany (MSC).
authors.forename: Matteo
authors.initials: M
authors.lastname: Setti
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Savalli
authors.forename: Daniela
authors.initials: D
authors.lastname: Osti
authors.forename: Cristina
authors.initials: C
authors.lastname: Richichi
authors.forename: Marina
authors.initials: M
authors.lastname: Angelini
authors.forename: Paola
authors.initials: P
authors.lastname: Brescia
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Fornasari
authors.forename: Maria Stella
authors.initials: MS
authors.lastname: Carro
authors.forename: Michele
authors.initials: M
authors.lastname: Mazzanti
authors.forename: Giuliana
authors.initials: G
authors.lastname: Pelicci
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D063646:Carcinogenesis / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D051379:Mice; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation
chemical_list: C501966:CLIC1 protein, human; D018118:Chloride Channels; D034741:RNA, Small Interfering