HIF-1α is critical for hypoxia-mediated maintenance of glioblastoma stem cells by activating Notch signaling pathway

Abstract

Hypoxia induces the expansion of glioblastoma stem cells (GSCs), but the mechanism underlying it is still unclear. Here, we supply evidence that hypoxia-inducible factor-1α (HIF-1α) induced activation of Notch pathway is essential for hypoxia-mediated maintenance of GSC. Either depletion of HIF-1α or inactivation of Notch signaling partly inhibits the hypoxia-mediated maintenance of GSC. Further data suggest a role for HIF-1α in the interaction and stabilization of intracellular domain of Notch (NICD), and activation of Notch signaling. The mRNA level of HIF-1α and Notch target gene FABP7 was elevated in GSC. And the STAT3 pathway responsible for the HIF-1α gene transcription, the phosphatidylinositol 3-kinase-Akt and ERK1/2, both of which are contributed to HIF-1α protein translation, are also preferentially activated in GSC. Inhibition of these pathways partly reduces the hypoxia-induced activation of the Notch pathway and subsequent GSC maintenance. Taken together, our findings suggest that HIF-1α requires Notch pathway to drive the maintenance of GSC. The activated regulation of HIF-1α makes GSC more sensitive to hypoxia-mediated maintenance. These findings enhance our understanding of mechanism of hypoxia-mediated GSC expansion and provide HIF-1α as an attractive target for glioblastoma therapy.

Figure 1

Hypoxia facilitates the maintenance of GSC. (a) Expression of Nestin, GFAP in U251, SHG44, A172, and C6 glioblastoma cells cultured in hypoxia or normoxia. Bar=50 μm. (b and c) Glioblastoma cells were cultured in hypoxia for 36 h and then the colony-formation ability was investigated. The picture of colonies in a representative dish was taken with phase contrast microscopy. Bar=1 mm. (d) Cells exposed to a hypoxia for 36 h were fixed and then CD133-positive cells were analyzed by FACS. (e) Representative FACS SP analysis for glioblastoma cells showed that hypoxia resulted in increase in the ratio of SP cells. (f and g) Sphere-forming assay showed that the self-renewal capacity was increased when incubated at hypoxia (2% O₂) compared with normoxia (20% O₂). The picture of cells in a representative well was taken with phase contrast microscopy. Bar=200 μm. The results shown in the graph are mean±S.D. from three experiments. Significance of difference is indicated as ^*P<0.05, ^**P<0.01

Figure 2

Elevated HIF-1α and activation of Notch pathway in U251SC. (a) Some differentially expressed genes found in microarray analysis were listed. (b) RT-PCR analysis for some differentially expressed genes. (c) Expression levels of protein in U251 and U251SC were analyzed by two-dimensional gel electrophoresis. (d) Some differently expressed proteins that are related to cancer and stem cell were listed. (e) Proteins indicated by red arrow, including FABP7, TPT1, ERP57 and HSPB1 were selected for validation by western blot assay. Data are from representative experiments repeated at least three times. The color reproduction of this figure is available at the Cell Death and Differentiation journal online.

Figure 3

Hypoxia requires notch pathway to drive the maintenance of U251 cells. (a) Pretreatment with Notch1 siRNA, Delta1 siRNA, or γ-secretase inhibitor (DAPT; 20 μM) for 4 h abrogate the hypoxia-induced expansion of Nestin-positive cells. (b) U251 cells were treated with hypoxia for 24 h in the presence or absence of Notch1 siRNA, Delta1 siRNA, or DAPT (20 μM), the expression of Notch1, Delta1, and Notch target genes such as FABP7 and Hey1 were then analyzed by RT-PCR. The expression of NICD, Delta1, Notch1, and FABP7 proteins was analyzed by western blot assay after treated with hypoxia for 36 h. (c) Pretreatment with HIF-1α siRNA, or DAPT (20 μM) for 4 h abrogate the hypoxia and CoCl₂ (100 μM)-induced expansion of Nestin-positive cells. Bar=50 μm. (d) U251 cells were treated with hypoxia and CoCl₂ (100 μM) for 24 h in the presence or absence of HIF-1α siRNA and DAPT (20 μM), the expression of HIF-1α, PGK1, and Notch target genes such as Hey1, Hes1, FABP7 were then analyzed by RT-PCR. The expression of NICD, FABP7, and HIF-1α protein in U251 cells treated with hypoxia and CoCl₂ (100 μM) for 36 h in the presence or absence of HIF-1α siRNA and DAPT (20 μM) was analyzed by western blot assay after treated with hypoxia for 36 h

Figure 4

HIF-2α is not essential for hypoxia-mediated maintenance of U251 cells. (a) Expression of HIF-2α mRNA in U251 and U251SC either at normoxia or hypoxia for 24 h was evaluated by RT-PCR. (b) Expression of HIF-2α and PHD1/2/3 in U251 and U251SC either at normoxia or hypoxia for 36 h was evaluated. (c) Pretreatment with HIF-2α siRNA abrogate the hypoxia-induced increase of HIF-2α protein. (d) Pretreatment with HIF-2α siRNA could not abrogate the hypoxia-induced expansion of Nestin-positive cells. Bar=50 μm. (e) Pretreatment with HIF-2α siRNA could not abrogate the hypoxia-induced increase of colony-formation ability in U251 cells. The picture of colonies in a representative dish was taken with phase contrast microscopy. Bar=1 mm. (f) Representative FACS dot plot analysis for glioma cells showed that HIF-2α siRNA could not abrogate the hypoxia-induced increase in the number of CD133-positive cells. Cells exposed to hypoxia for 36 h were fixed and analyzed by FACS. Data are from representative experiments repeated at least three times

Figure 5

HIF-1α activates Notch signal by interacting with and stabilizing NICD. (a) The expression of endogenous HIF-1α and NICD in U251 and U251SC exposed to hypoxia was analyzed. (b) HIF-1α was immunoprecipitated and subjected to SDS-PAGE. (c) The expression of HIF-1α and some genes involved in Notch signaling in U251 and U251SC exposed to hypoxia was analyzed

Figure 6

Hypoxia requires PI3K/AKT, ERK, and STAT3 signaling to drive the maintenance of U251 cells. (a) PI3K/AKT, ERK/MAPK, and STAT3 signaling pathways were preferentially activated in U251SC. (b) U251 cells were treated with hypoxia and CoCl₂ (100 μM) for 24 h in the presence or absence of rapamycin (10 μM), PD98059 (20 μM), and WP1066 (10 μM), PGK1 and Notch target genes such as Hey1, Hes1, FABP7 were then analyzed by RT-PCR. The expression levels of NICD, FABP7, and HIF-1α protein were analyzed by western blot assay after treated with hypoxia for 36 h. (c) U251 cells were treated with hypoxia and CoCl₂ (100 μM) for 24 h in the presence or absence of siAKT, siERK2 and siSTAT3, PGK1 and Notch target genes such as Hey1, Hes1, FABP7 were then analyzed by RT-PCR. The expression levels of NICD, FABP7, and HIF-1α protein were analyzed by western blot assay after treated with hypoxia for 36 h. (d and e) Pretreatment with mTOR inhibitor (rapamycin; 10 μM), ERK inhibitor (PD98059; 20 μM), STAT3 inhibitor (WP1066; 10 μM), and indicated siRNA for 4 h abrogate the hypoxia and CoCl₂ (100 μM) induced expansion of Nestin-positive cells. Bar=50 μm

Figure 7

Hypoxia facilitates the maintenance of primary glioblastoma stem cells in a HIF1α and Notch signal-dependent manner. (a, b and c). Compared with control, significance of difference is indicated as ^*P<0.05. Pretreatment with siRNA of Notch1, HIF-1α, ERK2, AKT, and STAT3 for 4 h abrogated the hypoxia-induced expansion of Nestin-positive cells, CD133-positive cells, and number of spheres. Bar=50 μm. (d) Cells were treated with hypoxia for 24 h in the presence or absence of indicated siRNA, the expression of Notch1, HIF-1α, and FABP7 mRNA were then analyzed by RT-PCR. The expression levels of Notch1, NICD, HIF-1α, and FABP7 protein were analyzed by western blot assay after treated with hypoxia for 36 h

Figure 8

Schematic depiction of crosstalk between Notch and HIF-1α. (1) The canonical hypoxic signaling leads to the inhibition of HIF-1α degradation mediated by PHD1/2/3. STAT3 could bind to the HIF-1α promoter and stimulates its transcription, whereas PI3K/AKT, ERK were contributed to the translation of HIF-1α protein through phosphorylation of the eukaryotic translation initiation factor 4E (eIF-4E) binding protein (4E-BP1) and p70 S6 kinase (S6K). Elevation of HIF-1α protein induces the activation of downstream genes, such as VEGF, PGK1, and ET1, through binding to HRE elements in the corresponding promoters. (2) After binding to ligand, the Notch receptor undergoes two proteolytic cleavages and then releases the intracellular domain of NICD, which translocates to the nucleus and associates with the CSL transcription factor and coactivators such as RPBJK, and regulates the expression of target genes such as Hey and FABP7. (3) HIF-1α and NICD form a point of convergence between the two signaling, leading to stabilization of NICD, recruitment of HIF-1α to Notch-responsive promoters, and activation of Notch downstream genes. HIF-1α could also induce the elevation of DLL1, which may contribute to HIF-1α-mediated activation of Notch pathway. 1, the left part; 2, the right part; 3, the central part