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Functional role of CLIC1 ion channel in glioblastoma-derived stem/progenitor cells.

Setti M, Savalli N, Osti D, Richichi C, Angelini M, Brescia P, Fornasari L, Carro MS, Mazzanti M, Pelicci G - J. Natl. Cancer Inst. (2013)

Bottom Line: CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment.The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient-derived neurospheres.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.

View Article: PubMed Central - PubMed

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).

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.

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Effect of chloride intracellular channel 1 (CLIC1) silencing in glioblastoma (GBM) neurospheres. A) Western blotting analysis showing the efficiency of CLIC1 silencing in GBM neurospheres isolated from four patient samples. Dissociated neurospheres were transduced with lentivirus carrying either nontargeting short-hairpin RNA (shRNA) (NT) or CLIC1 shRNA (sh). Mouse embryonic fibroblasts derived from CLIC1 knockout mice (MEF CLIC1-KO) were used as negative controls. Vinculin was used as loading control. B and D) Representative current traces (total, indanyloxyacetic acid 94 [IAA94]–sensitive, and 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid [DIDS]–sensitive currents) from NT (B) and CLIC1-silenced (sh) (D) cells derived from GBM patient 10 (hGBM#10) NS and elicited by different potential steps (from −60 mV to 60 mV). C and E) The current–voltage relationships for the corresponding experiments in B and D. F) CLIC1-sensitive currents (IIAA94) were isolated using the specific CLIC1 inhibitor IAA94, and normalized to the total cell current (ITot) (IIAA94/ITot%). G) The other chloride (Cl−) currents in the cells were evaluated by the inhibitor DIDS (IDIDS) and normalized to the total cell current (ITot) (IDIDS/ITot%). Mean values derived from four independent experiments are represented. Error bars represent 95% confidence intervals. GLM test of between-subjects effects on IIAA94/ITot values: F for “potential” = 0.108, d. f. = 4, p = 0.979 (n. s.); F for “cell type” = 50.038, d. f. = 1, p < 0.001; F for variables interaction = 0.058, d. f. = 4, p = 0.993 n. s. No significance for interaction means a similar pattern of IIAA94/ITot change for different cell types at different membrane potential values, even if mean IIAA94/ITot values are different between different cell types. Generalized linear model test of between-subjects effects on IDIDS / ITot values: F for “potential” = 4.031, degrees of freedom (df) = 4, P = .01; F for “cell type” = 3.590, df = 1, P = .07; F for variables interaction = 0.085, df = 4, P = .99. No statistical significance for interaction means a similar pattern of IDIDS/ITot change for different cell types at different membrane potential values.
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Figure 4: Effect of chloride intracellular channel 1 (CLIC1) silencing in glioblastoma (GBM) neurospheres. A) Western blotting analysis showing the efficiency of CLIC1 silencing in GBM neurospheres isolated from four patient samples. Dissociated neurospheres were transduced with lentivirus carrying either nontargeting short-hairpin RNA (shRNA) (NT) or CLIC1 shRNA (sh). Mouse embryonic fibroblasts derived from CLIC1 knockout mice (MEF CLIC1-KO) were used as negative controls. Vinculin was used as loading control. B and D) Representative current traces (total, indanyloxyacetic acid 94 [IAA94]–sensitive, and 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid [DIDS]–sensitive currents) from NT (B) and CLIC1-silenced (sh) (D) cells derived from GBM patient 10 (hGBM#10) NS and elicited by different potential steps (from −60 mV to 60 mV). C and E) The current–voltage relationships for the corresponding experiments in B and D. F) CLIC1-sensitive currents (IIAA94) were isolated using the specific CLIC1 inhibitor IAA94, and normalized to the total cell current (ITot) (IIAA94/ITot%). G) The other chloride (Cl−) currents in the cells were evaluated by the inhibitor DIDS (IDIDS) and normalized to the total cell current (ITot) (IDIDS/ITot%). Mean values derived from four independent experiments are represented. Error bars represent 95% confidence intervals. GLM test of between-subjects effects on IIAA94/ITot values: F for “potential” = 0.108, d. f. = 4, p = 0.979 (n. s.); F for “cell type” = 50.038, d. f. = 1, p < 0.001; F for variables interaction = 0.058, d. f. = 4, p = 0.993 n. s. No significance for interaction means a similar pattern of IIAA94/ITot change for different cell types at different membrane potential values, even if mean IIAA94/ITot values are different between different cell types. Generalized linear model test of between-subjects effects on IDIDS / ITot values: F for “potential” = 4.031, degrees of freedom (df) = 4, P = .01; F for “cell type” = 3.590, df = 1, P = .07; F for variables interaction = 0.085, df = 4, P = .99. No statistical significance for interaction means a similar pattern of IDIDS/ITot change for different cell types at different membrane potential values.

Mentions: To disclose the role of CLIC1 in GBM stem/progenitor cells, we silenced CLIC1 expression in patient-derived GBM neurospheres by cloning short-hairpin RNA (shRNA) oligonucleotides specific against human CLIC1 mRNA (sh) in a lentiviral vector containing green fluorescent protein and the puromycin resistance gene as reporters. The same vector containing an shRNA targeting the luciferase mRNA sequence was used as control (nontargeting [NT]). Interference efficiency was confirmed by western blot: CLIC1 was silenced by nearly 90% in different samples (Figure 4A; Supplementary Figure 4A, available online).


Functional role of CLIC1 ion channel in glioblastoma-derived stem/progenitor cells.

Setti M, Savalli N, Osti D, Richichi C, Angelini M, Brescia P, Fornasari L, Carro MS, Mazzanti M, Pelicci G - J. Natl. Cancer Inst. (2013)

Effect of chloride intracellular channel 1 (CLIC1) silencing in glioblastoma (GBM) neurospheres. A) Western blotting analysis showing the efficiency of CLIC1 silencing in GBM neurospheres isolated from four patient samples. Dissociated neurospheres were transduced with lentivirus carrying either nontargeting short-hairpin RNA (shRNA) (NT) or CLIC1 shRNA (sh). Mouse embryonic fibroblasts derived from CLIC1 knockout mice (MEF CLIC1-KO) were used as negative controls. Vinculin was used as loading control. B and D) Representative current traces (total, indanyloxyacetic acid 94 [IAA94]–sensitive, and 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid [DIDS]–sensitive currents) from NT (B) and CLIC1-silenced (sh) (D) cells derived from GBM patient 10 (hGBM#10) NS and elicited by different potential steps (from −60 mV to 60 mV). C and E) The current–voltage relationships for the corresponding experiments in B and D. F) CLIC1-sensitive currents (IIAA94) were isolated using the specific CLIC1 inhibitor IAA94, and normalized to the total cell current (ITot) (IIAA94/ITot%). G) The other chloride (Cl−) currents in the cells were evaluated by the inhibitor DIDS (IDIDS) and normalized to the total cell current (ITot) (IDIDS/ITot%). Mean values derived from four independent experiments are represented. Error bars represent 95% confidence intervals. GLM test of between-subjects effects on IIAA94/ITot values: F for “potential” = 0.108, d. f. = 4, p = 0.979 (n. s.); F for “cell type” = 50.038, d. f. = 1, p < 0.001; F for variables interaction = 0.058, d. f. = 4, p = 0.993 n. s. No significance for interaction means a similar pattern of IIAA94/ITot change for different cell types at different membrane potential values, even if mean IIAA94/ITot values are different between different cell types. Generalized linear model test of between-subjects effects on IDIDS / ITot values: F for “potential” = 4.031, degrees of freedom (df) = 4, P = .01; F for “cell type” = 3.590, df = 1, P = .07; F for variables interaction = 0.085, df = 4, P = .99. No statistical significance for interaction means a similar pattern of IDIDS/ITot change for different cell types at different membrane potential values.
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Figure 4: Effect of chloride intracellular channel 1 (CLIC1) silencing in glioblastoma (GBM) neurospheres. A) Western blotting analysis showing the efficiency of CLIC1 silencing in GBM neurospheres isolated from four patient samples. Dissociated neurospheres were transduced with lentivirus carrying either nontargeting short-hairpin RNA (shRNA) (NT) or CLIC1 shRNA (sh). Mouse embryonic fibroblasts derived from CLIC1 knockout mice (MEF CLIC1-KO) were used as negative controls. Vinculin was used as loading control. B and D) Representative current traces (total, indanyloxyacetic acid 94 [IAA94]–sensitive, and 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid [DIDS]–sensitive currents) from NT (B) and CLIC1-silenced (sh) (D) cells derived from GBM patient 10 (hGBM#10) NS and elicited by different potential steps (from −60 mV to 60 mV). C and E) The current–voltage relationships for the corresponding experiments in B and D. F) CLIC1-sensitive currents (IIAA94) were isolated using the specific CLIC1 inhibitor IAA94, and normalized to the total cell current (ITot) (IIAA94/ITot%). G) The other chloride (Cl−) currents in the cells were evaluated by the inhibitor DIDS (IDIDS) and normalized to the total cell current (ITot) (IDIDS/ITot%). Mean values derived from four independent experiments are represented. Error bars represent 95% confidence intervals. GLM test of between-subjects effects on IIAA94/ITot values: F for “potential” = 0.108, d. f. = 4, p = 0.979 (n. s.); F for “cell type” = 50.038, d. f. = 1, p < 0.001; F for variables interaction = 0.058, d. f. = 4, p = 0.993 n. s. No significance for interaction means a similar pattern of IIAA94/ITot change for different cell types at different membrane potential values, even if mean IIAA94/ITot values are different between different cell types. Generalized linear model test of between-subjects effects on IDIDS / ITot values: F for “potential” = 4.031, degrees of freedom (df) = 4, P = .01; F for “cell type” = 3.590, df = 1, P = .07; F for variables interaction = 0.085, df = 4, P = .99. No statistical significance for interaction means a similar pattern of IDIDS/ITot change for different cell types at different membrane potential values.
Mentions: To disclose the role of CLIC1 in GBM stem/progenitor cells, we silenced CLIC1 expression in patient-derived GBM neurospheres by cloning short-hairpin RNA (shRNA) oligonucleotides specific against human CLIC1 mRNA (sh) in a lentiviral vector containing green fluorescent protein and the puromycin resistance gene as reporters. The same vector containing an shRNA targeting the luciferase mRNA sequence was used as control (nontargeting [NT]). Interference efficiency was confirmed by western blot: CLIC1 was silenced by nearly 90% in different samples (Figure 4A; Supplementary Figure 4A, available online).

Bottom Line: CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment.The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient-derived neurospheres.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.

View Article: PubMed Central - PubMed

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).

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.

Show MeSH
Related in: MedlinePlus