Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells

Abstract

Glioblastomas are lethal cancers characterized by florid angiogenesis promoted in part by glioma stem cells (GSCs). Because hypoxia regulates angiogenesis, we examined hypoxic responses in GSCs. We now demonstrate that hypoxia-inducible factor HIF2alpha and multiple HIF-regulated genes are preferentially expressed in GSCs in comparison to non-stem tumor cells and normal neural progenitors. In tumor specimens, HIF2alpha colocalizes with cancer stem cell markers. Targeting HIFs in GSCs inhibits self-renewal, proliferation, and survival in vitro, and attenuates tumor initiation potential of GSCs in vivo. Analysis of a molecular database reveals that HIF2A expression correlates with poor glioma patient survival. Our results demonstrate that GSCs differentially respond to hypoxia with distinct HIF induction patterns, and HIF2alpha might represent a promising target for antiglioblastoma therapies.

Figure 1. Glioblastoma Stem and Nonstem Cells Differentially Expressed Hypoxia Response Genes

Glioblastoma stem and nonstem isolated from the glioblastoma xenograft D456MG were treated with desferrioxamine (DFX) to mimic hypoxia for the time indicated. RT-PCR was performed with primers specific for HIF2α (A), HIF1α (B), Oct4 (C), phosphoglycerate kinase 1, PGK1 (D), glucose transporter type 1, Glut1 (E), transforming growth factor alpha, TGF-α (F), SerpinB9 (G), and vascular endothelial growth factor, VEGF (H). Data were normalized to GAPDH, Ubiquitin C, and SDHA. #, P < 0.01 with ANOVA comparison of stem cells to nonstem cells with identical treatments. *, P < 0.01 with ANOVA comparison of stem cells under hypoxia vs. normoxia. ≈, P < 0.01 with ANOVA comparison of on-stem cells under hypoxia vs. normoxia.

Figure 2. Glioma Stem Cells and Normal Neural Progenitors Differentially Expressed Hypoxia Response Genes

Cells from T3565 and T4302 glioblastoma samples and two different normal neural progenitor cell preparations were cultured in normoxia (20% oxygen) or hypoxia (1% oxygen) for 24 hr. RT-PCR analysis was performed with primers specific for HIF2α (A), HIF1α (B), Oct4 (C), PGK1 (D), Glut1 (E), TGF-α (F), SerpinB9 (G), and VEGF (H). Data were normalized to β-actin levels. *, P < 0.01 with ANOVA comparison of hypoxia treated cells to identically prepared normoxia controls. #, P < 0.01 with ANOVA comparison of indicated hypoxia treated cells to both hypoxia treated GSCs.

Figure 3. Hypoxia Potently Induced HIF2α Protein Expression in Glioma Stem Cells

(A-G) Glioblastoma stem and nonstem cells isolated from multiple samples were treated with DFX to mimic hypoxia for the indicated times. Nuclear and cytoplasmic fractions (B) or total cell lysate (A, C, E-G) were analyzed. (H) Cells isolated from D456MG were cultured under 20% or 0.2% oxygen for 24 hr and analyzed by immunoblotting. (I) Cells isolated from TB080076 were cultured under normoxia (20% oxygen) or hypoxia (1% oxygen) for 24 hr and analyzed by immunoblotting. (J) Cells isolated from T3359 were cultured under more modest hypoxia (2% oxygen) and total cell lysates were analyzed. (K) T3359 GSCs and CD133+ normal neural progenitors were treated with 100 μM DFX and total cell lysates were analyzed. (L) GSCs (T4302, T3691, and T3946) and the normal human neural progenitors (16399 and 16529) were treated with DFX and analyzed by immunoblotting. (M) Higher HIF2α protein expression is maintained with a relatively physiological level of oxygen in GSCs. Cells were cultured under physiological level of oxygen (5% oxygen) for the indicated times and total cell lysates collected. In (J) and (M), and DFX treated samples were used as a positive control.

Figure 4. HIF2α Co-Expressed with Cancer Stem Cell Markers in Human Glioblastoma Biopsy Specimens

(A) Restricted pattern of HIF2α and stem cell marker expression in human brain tumor patient specimens. (B) Immunofluorescence of cells in human brain tumor patient specimens demonstrates co-localization of CD133 and HIF2α. (C-D) Cells expressing the cancer stem cell marker CD133 also express HIF2α. Glioma stem and nonstem cells isolated from T3691 (C) or T3359 (D) were analyzed for CD133 and HIF2α expression via FACS using anti-CD133-APC and anti-HIF2α-PE.

Figure 5. HIF Knockdown Altered Glioma Stem Cell Neurosphere Formation

(A) Specific knockdown of HIF1α and HIF2α protein using shRNA. (B-E) 1, 10, or 100 lentiviral infected GSCs isolated from T3359 were cultured in 24-well plates. (B) Representative images of spheres are shown. (C) The total number of neurospheres per well is significantly decreased with HIF targeting. (D) Neurosphere size is significantly reduced by targeting HIF expression. (E) Representative image of neurospheres of neurosphere size in D. (F-G) Targeting HIFs in T3359 or T3832 stem cells decreases neurosphere formation in sequential passages. (H) Representative images of neurospheres formed in sequential passage neurosphere formation assays. *, P < 0.001

Figure 6. HIF Knockdown Reduced Glioma Stem Cell Growth Due to Elevated Apoptosis

(A) Targeting HIF2α in glioblastoma stem, but not nonstem, cells decreases growth. Cell titers were determined using the CellTiter-Glo Luminescent Cell Viability Assay kit. *, P < 0.05 with ANOVA comparison of HIF1α shRNA to either NT shRNA or HIF2α shRNA. #, P <0.01 with ANOVA comparison of NT shRNA to either HIF1α or HIF2α shRNA. (B) Cells were plated and quantified as in A but cultured in 20% or 2% oxygen as indicated. Inset shows effective knockdown with HIF2α and HIF1α shRNAs. (C-D) Targeting HIFs leads to increased apoptosis in GSCs as determined by the Annexin V staining. *, P < 0.05 with ANOVA comparison to nontargeting shRNA of the same cell type and hypoxia treatment. #, P <0.01 with ANOVA comparison of HIF2α shRNA treated GSCs to HIF2α shRNA treated nonstem cells with identical oxygen treatment. (E) Targeting HIFs results in increased caspase-3/7 activity in GSCs. *, P < 0.05

Figure 7. HIF Knockdown Decreased Glioma Stem Cell Mediated Angiogenesis

(A) HIF2α knockdown prevents hypoxia induced activation of the VEGF Promoter. *, P < 0.001 with ANOVA comparison of HIF2α or HIF1α shRNA treated stem cells to nontargeting shRNA with hypoxia. (B) HIF knockdown reduces VEGF protein expression in GSCs. (C) HIF2α knockdown reduces VEGF expression in stem, but not nonstem, glioblastoma cells. *, P < 0.01 (D-E) HIF knockdown reduces VEGF mRNA level in GSCs. *, P < 0.01 (F-H) HIF2α knockdown in GSCs reduced cancer cell-mediated endothelial cell proliferation. (F) Representative diagram of the coculture assay. (G) Representative images of cocultured HMVEC cells after cells fixed with 4% PFA and then stained with toluidine blue. (H) HMVEC proliferation was measured through [3H]-thymidine incorporation. *, P < 0.001 by ANOVA with comparison of HIF2α or HIF1α shRNA infected glioma cells to corresponding nontargeting shRNA infected cells. #, P < 0.001 by ANOVA comparison of nontargeting shRNA infected GSCs to nonstem nontargeting control cells.

Figure 8. HIF Knockdown Suppressed Cancer Stem Cell Mediated Tumor Growth

(A) Gross histology demonstrates highly vascular tumors in glioblastoma stem derived tumors from cells infected with nontargeting shRNA but not HIF targeting shRNAs. (B) Targeting VEGF or HIFs within the cancer stem cell subpopulation increases survival. 5000 infected GSCs were mixed with 95000 matched uninfected nonstem cells and injected into the mice brains. *, P < 0.03. (C-E) Targeting HIFs in the cancer stem cell subpopulation decreases tumorigenesis. *, P < 0.05. (F) Tumors do not form from GSCs selected for the incorporation of a puromycin marker associated with HIF targeting shRNA. *, P < 0.03. (G) In vivo limiting dilution assay demonstrates GSCs are less tumorigenic when HIFs are targeted. *, P < 0.03. (H) HIF2α but not HIF1α mRNA level correlates with patient survival (the Rembrandt database of the National Cancer Institute). There was only one patient with more than two fold HIF2A downregulation. No patients with HIF1A downregulation were observed. Analysis of the NCI TCGA database yields similar results (data not shown).