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, 28 (11), 1918-29

Interaction of Hypoxia-Inducible factor-1α and Notch Signaling Regulates Medulloblastoma Precursor Proliferation and Fate

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Interaction of Hypoxia-Inducible factor-1α and Notch Signaling Regulates Medulloblastoma Precursor Proliferation and Fate

Francesca Pistollato et al. Stem Cells.

Abstract

Medulloblastoma (MDB) is the most common brain malignancy of childhood. It is currently thought that MDB arises from aberrantly functioning stem cells in the cerebellum that fail to maintain proper control of self-renewal. Additionally, it has been reported that MDB cells display higher endogenous Notch signaling activation, known to promote the survival and proliferation of neoplastic neural stem cells and to inhibit their differentiation. Although interaction between hypoxia-inducible factor-1α (HIF-1α) and Notch signaling is required to maintain normal neural precursors in an undifferentiated state, an interaction has not been identified in MDB. Here, we investigate whether hypoxia, through HIF-1α stabilization, modulates Notch1 signaling in primary MDB-derived cells. Our results indicate that MDB-derived precursor cells require hypoxic conditions for in vitro expansion, whereas acute exposure to 20% oxygen induces tumor cell differentiation and death through inhibition of Notch signaling. Importantly, stimulating Notch1 activation with its ligand Dll4 under hypoxic conditions leads to expansion of MDB-derived CD133(+) and nestin(+) precursors, suggesting a regulatory effect on stem cells. In contrast, MDB cells undergo neuronal differentiation when treated with γ-secretase inhibitor, which prevents Notch activation. These results suggest that hypoxia, by maintaining Notch1 in its active form, preserves MDB stem cell viability and expansion.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

Figures

Figure 1
Figure 1. 2% oxygen preserves viability and proliferation of MDB derived cells
(A) representative pictures of HuTu33 cells expanded at 2%, 5% or 20% oxygen for 7 days. (B,C) Total cell number counts (by trypan blue exclusion) of MDB (B) and SVZ cells (HuSC30 and HuSC23) (C) cultured for 7 days. For (B), mean of 5 different MDB ± S.E.M., n = 2 for each tumor; for (C), mean of 2 different normal SVZ cells cultures ± S.E.M., n = 4 for each cell culture. 10X pictures, bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2. Acute exposure to high oxygen tension promotes neuronal differentiation, mitotic arrest and eventually cell death in MDB derived cells
(A) Representative immunocytochemical images (HuTu33) of (A) Ki67 (red)/cleaved-Caspase3 (green), (B) p21cip1 (red), (C) nestin (green)/β-III-tubulin (red), (D) nestin (green)/GFAP (red) and (E) nestin (green)/MAP2 (red) staining of MDB cells expanded for 2 days at either 2% or 20% oxygen. Quantifications in (F,G); for all graphs, mean of 5 different MDB ± S.E.M., n = 3 for each tumor. (H) QRT-PCR analyses of Sox2, EN1, Math1, Neurod1, NSCL1, β-III-tubulin, Pax6 and MAP2, normalized to gusb and then calibrated to 2% oxygen control. Mean ± S.E.M of 4 different MDB. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.
Figure 3
Figure 3. Acute exposure to high oxygen tension promotes Notch1 pathway inhibition and HIF-1α is required to maintain Notch1 signaling activated
(A) Representative western blot analyses of HIF-1α, Notch1, NICD, Dll4, Hes1, β-III-tubulin and β-actin of MDB cells acutely exposed to 20% oxygen for 30, 60 or 120 minutes. (B) Notch1-Luciferase transfected MDB cells, have been either left at 2% oxygen or acutely exposed to 20% oxygen for 8 hrs. The graph reports mean of 3 different MDB. (C) Representative western blot analyses of cells that had been transduced with a siHIF-1α/EGFP or si-LUC/EGFP bearing vectors. (D,E) Representative immunohistochemical images of (D) HIF-1α (red)/β-III-tubulin (green), (E) HIF-1α (red)/nestin (green) staining of MDB tissues. See quantification in Suppl. Table 2. Magnification 10X, bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001. RLU, relative light units; v, vessels; MVD, microvessel density.
Figure 4
Figure 4. Notch1, Hes1 and HIF-1α are co-expressed in nestin+ and CD133+ MDB cells in vivo
Representative immunohistochemical images of (A) HIF-1α (red)/Notch1 (green), (B) HIF-1α (red)/Hes1 (green), (C) nestin (red)/Notch1 (green), (D) HIF-1α (red)/CD133 (green) and (E) HIF-1α (red)/nestin (green) staining of MDB tissues. Insets in panel A, B show nuclear localization of Notch1 (left, A), HIF-1α (middle, A, B and E), and Hes1 (left, B) and cytoplasmic localization of nestin (left, E). 10X pictures, bar = 100 μm (for A,B,C,D,E), 60X insets, bar = 20 μm (in A,B merge). See quantification in Table 1.
Figure 5
Figure 5. Effects of Notch1 signaling exogenous modulation by Dll4 and DAPT on MDB cells
(A) Representative western blot analyses of HIF-1α, PHD2, FKBP38, Notch1, NICD, Dll4, Hes1, β-III-tubulin, p21cip1, PARP and β-actin of MDB derived cells cultured for 72 hrs in presence of either immobilized Dll4 (2 μg/ml) or DAPT (10 μM) at either 2% or 20% oxygen. (B) HRE-luciferase transfected cells in the same conditions as in (A) at 2% oxygen. (C) Notch1-luciferase transfected MDB cells treated with Dll4 and DAPT for 8 hr as described in Materials and Methods. For both B and C, values are expressed in RLU (= relative light units). (D) QRT-PCR analysis of hes1 normalized to gusb and then calibrated to 2% oxygen control. Mean ± S.E.M of 3 different MDB. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6. Notch1 signaling modulation by Dll4 and DAPT affects MDB cell phenotype depending on hypoxia
(A) Percentages of CD133+ cells of MDB derived cells cultured for 72 hr in presence of either immobilized Dll4 (2 μg/ml) or DAPT (10 μM) at either 2% or 20% oxygen. Mean of 3 tumors ± S.E.M., n = 2 for each tumor. (B-E) Immunocytochemical analysis for nestin (B) β-III-tubulin (C), Ki67 (D) and total cell quantification relative to total DAPI+ cells (E). MDB cells were treated as described in (A). Mean of 6 tumors ± S.E.M. (F) Representative immunocytochemical images of nestin (green)/β-III-tubulin (red) staining. 20X pictures, bar = 40 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

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