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. 2016 Oct 25;7(43):69173-69187.
doi: 10.18632/oncotarget.12507.

IKK/NF-κB Signaling Contributes to Glioblastoma Stem Cell Maintenance

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Free PMC article

IKK/NF-κB Signaling Contributes to Glioblastoma Stem Cell Maintenance

Amanda L Rinkenbaugh et al. Oncotarget. .
Free PMC article

Abstract

Glioblastoma multiforme (GBM) carries a poor prognosis and continues to lack effective treatments. Glioblastoma stem cells (GSCs) drive tumor formation, invasion, and drug resistance and, as such, are the focus of studies to identify new therapies for disease control. Here, we identify the involvement of IKK and NF-κB signaling in the maintenance of GSCs. Inhibition of this pathway impairs self-renewal as analyzed in tumorsphere formation and GBM expansion as analyzed in brain slice culture. Interestingly, both the canonical and non-canonical branches of the NF-κB pathway are shown to contribute to this phenotype. One source of NF-κB activation in GBM involves the TGF-β/TAK1 signaling axis. Together, our results demonstrate a role for the NF-κB pathway in GSCs and provide a mechanistic basis for its potential as a therapeutic target in glioblastoma.

Keywords: NF-κB; cancer stem cells; glioblastoma; tumor-initiating cells.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors state no conflict of interest.

Figures

Figure 1
Figure 1. NF-κB is preferentially activated in CD133+ glioblastoma stem cells
A. MTS assay using normal neural stem cells, astrocytes, or two GBM explants: 6969 and 7030. Cells were treated daily with DMSO or 5 μM Compound A and analyzed every 24 hours for 96 hours. Data are represented as the mean ± s.d. and are representative of three independent experiments. B. Analysis of total cells, isolated CD133-, or CD133+ cells by immunoblot for phosphorylation of p65. Quantification of the immunoblots for the ratio of phospho-p65 to total p65 is: 7063 total: 1; CD133-: 0.23; CD133+: 1.77; 7030 total: 1; CD133-: 0.19; CD133+: 1.15.
Figure 2
Figure 2. Pharmacological inhibition of the IKK/NF-κB pathway decreases tumorsphere formation
7030 CD133+ cells plated for a tumorsphere assay and treated as indicated (DMSO or 5μM Compound A, once or daily). After one week of growth, tumorsphere formation was analyzed. A. Representative images of tumorsphere formation after daily treatment. B. Quantification of primary tumorspheres formed per well for two explants. Data are represented as mean ± SEM, ****p < 0.0001, ***p < 0.001, *p < 0.05 by t-test. C. Primary tumorspheres were dissociated, replated, and treated again as indicated. Secondary tumorsphere formation was quantified after another week of growth. Data are represented as mean ± SEM, ****p < 0.0001, ***p < 0.001, *p < 0.05 by t-test. D., E. Tumorsphere formation was measured through a limiting dilution assay with 7030 D. or GBM6 E. CD133+ cells plated at 100, 50, 20, 10, 5, or 1 cell(s)/well and treated with DMSO or 5μM Compound A (7030: n = 48 wells/condition; p = 2.02×10−47; GBM6: n≥116wells/condition; p = 2.23×10−11).
Figure 3
Figure 3. Genetic inhibition of the IKK/NF-κB pathway decreases tumorsphere formation
7030 CD133+ cells were transfected with siRNA (control, p65, or IKKβ) and then plated for tumorsphere assays A. Quantification of tumorspheres formed per well after one week of growth. Data are represented as the mean ± SEM, ****p < 0.0001 by t-test. B. Transfected cells analyzed by immunoblot for p-p65, p65, IKKβ, and β-tubulin. C. Representative images of tumorsphere formation after daily treatment. D. Tumorsphere formation was measured through a limiting dilution assay with GBM6 CD133+ cells plated at 100, 50, 20, 10, 5, or 1 cell(s)/well following transfection with control or p65 siRNA (n≥70 wells; p = 1.76×10−6).
Figure 4
Figure 4. Multiple NF-κB subunits contribute to tumorsphere formation
A. GBM6 CD133+ cells were transfected with siRNA control or targeting p65, RelB, or p100. Subsequently, cells were plated out for limiting dilution assay at 100, 50, 20, 10, 5, or 1 cell(s)/well and scored for the presence or absence of tumorspheres following one week of growth (n≥95 wells/condition; p65 vs. control p = 2.26×10−5; RelB vs. control p = 6.44x10−5; p100 vs. control p = 3.82x10−5). B. Quantitative real-time PCR was performed to analyze expression of RELA, RELB, or NFKB2 in siRNA-transfected cells normalized to GUSB expression. C. Expression of canonical and non-canonical NF-κB components was analyzed by immunoblot in 7030 CD133+ cells transfected with siRNA (control, p65, or IKKβ). D. GBM6 CD133+ cells were transfected with siRNA (control, IKKα, or IKKβ). Subsequently, cells were plated out for limiting dilution assay at 50, 20, 10, 5, 2, or 1 cell(s)/well and scored for the presence or absence of tumorspheres following one week of growth (n≥48 wells/condition; IKKα vs. control p = 0.192; IKKβ vs. control p = 6.01x10−5). E. Quantitative real-time PCR was performed to analyze expression of CHUK and IKBKB in siRNA-transfected cells normalized to GUSB expression.
Figure 5
Figure 5. TAK1 activates the NF-κB pathway to promote glioblastoma stem cell function
A. 6969 or 7030 cells were transfected with 3x-κB luciferase reporter, treated with the indicated inhibitors for 24 hours, then harvest and analyzed for luciferase activity (n = 3; **p < 0.0001 by t-test, error bars represent SEM) B. Quantification of tumorsphere formation in GBM6 CD133+ cells following treatment with either (5Z)-7-oxozeaenol or NG25 either once or daily. Data are represented as the mean ± SEM, **p < 0.0001 by t-test. C. Limiting dilution assay with GBM6 CD133+ cells transfected with siRNA control or TAK1 (n = 72 wells/condition; p < 0.05). D. Limiting dilution assay following treatment of GBM6 CD133+ cells with structurally distinct TAK1 inhibitors: 2.5 μM (5Z)-7-oxozeaenol or 2 μM NG-25 (n≥90wells/condition; 5Z vs. DMSO p = 1.01×10−19, NG vs. DMSO p = 5.29×10−10).
Figure 6
Figure 6. TGF-β is one source of NF-κB activation in GBM
A. 6969 and GBM6 explants were stimulated with 10ng/mL TGF-β for 6 or 24 hours, then analyzed by immunoblotting for phosphorylation of Smad and p65. B. 6969 and GBM6 explants were treated with 10 μM SB431542, a TGF-βR1 inhibitor, for 6 or 24 hours, then analyzed by immunoblotting for p65 phosphorylation. C. 6969 or 7030 cells were transfected with 3x-κB luciferase reporter and treated with DMSO or 10 μM SB431542 for 24 hours, then harvested and analyzed for luciferase activity (n = 3, **p < 0.0001, *p < 0.01 by t-test, error bars represent SEM). D. Quantification of tumorsphere formation in GBM6 CD133+ cells following treatment with 10 μM SB431542 either once or daily. Data are represented as the mean ± SEM, **p < 0.0001 by t-test.
Figure 7
Figure 7. Inhibition of the IKK/NF-κB pathway decreases glioblastoma growth/survival ex vivo.
A. Images of GFP+ GBM6 cells following siRNA transfection and brain slice culture. B. Average percent change in GFP+ area over the course of the experiment (n = 14 for control, 13 for p65; p < 0.005 by linear regression; error bars represent SEM) C. Images of GFP+ GBM6 cells over the course of eight days of culture on brain slices following treatment with DMSO or Compound A. D. Average percent change in GFP+ area over the course of the experiment (n = 12; p < 0.01 by linear regression; error bars represent SEM)

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