ZEB1 Promotes Invasion in Human Fetal Neural Stem Cells and Hypoxic Glioma Neurospheres

Abstract

Diffuse spread through brain parenchyma and the presence of hypoxic foci rimmed by neoplastic cells are two cardinal features of glioblastoma, and low oxygen is thought to drive movement of malignant gliomas in the core of the lesions. Transcription factors associated with epithelial-to-mesenchymal transition (EMT) have been linked to this invasion, and we found that hypoxia increased in vitro invasion up to fourfold in glioblastoma neurosphere lines and induced the expression of ZEB1. Immunohistochemical assessment of 295 surgical specimens consisting of various types of pediatric and adult brain cancers showed that ZEB1 expression was significantly higher in infiltrative lesions than less invasive tumors such as pilocytic astrocytoma and ependymoma. ZEB1 protein was also present in human fetal periventricular stem and progenitor cells and ZEB1 inhibition impaired migration of in vitro propagated human neural stem cells. The induction of ZEB1 protein in hypoxic glioblastoma neurospheres could be partially blocked by the HIF1alpha inhibitor digoxin. Targeting ZEB1 blocked hypoxia-augmented invasion of glioblastoma cells in addition to slowing them in normoxia. These data support the role for ZEB1 in invasive and high-grade brain tumors and suggest its key role in promoting invasion in the hypoxic tumor core as well as in the periphery.

Keywords: EMT; ZEB1; glioma; hypoxia; neural stem cell.

Figure 1

Immunohistochemical analysis of ZEB1 expression in brain tumors: ZEB1 protein was not detected in normal adult brain (A), with varying levels of nuclear staining in pilocytic astrocytoma (“PA”) (B), ependymoma (“EP”) (C), oligodendroglioma (“O”) (D), astrocytoma (“A”) (E), anaplastic astrocytoma (“AA”), adult GBM (“aGBM”) (F), pediatric GBM (“pGBM”) (G) and medulloblastoma (“MB”) (H). Analysis of tissue microarrays facilitated analysis of multiple cases from each group (I). Relatively solid tumors (pilocytic astrocytoma and ependymoma) had significantly lower levels of ZEB1 than infiltrative ones (J). Among infiltrating gliomas, Grade II tumors showed significantly lower ZEB1 expression than high‐grade ones (K) (original magnification 400× for A–H).

Figure 2

Hypoxia augmented invasion and activates ZEB1 in GBM‐derived neurospheres: Boyden chamber‐based cellular invasion is increased up to fourfold, representative light‐field microscope pictures of hematoxylin‐stained cells traversed the membrane in the two oxygen tension conditions (20× magnification) (A). Hypoxia increases expression of GMT activators ZEB1 and TWIST1 and SNAI1 (B + C). Strongly ZEB1‐positive cells in the cellular GBM core including a necrotic area (D, original magnification 400×). *P ≤ 0.05, **P ≤ 0.0001.

Figure 3

Targeting HIF1a inhibits ZEB1 and invasion: Digoxin‐mediated inhibition of HIF1a in GBM cells grown in hypoxia (A) shows inhibition of ZEB1 (B) accompanied with significant reduction of in vitro invasion (C). **P ≤ 0.0001.

Figure 4

Direct ZEB1 targeting inhibits invasion: shRNA‐mediated knockdown of ZEB1 as quantified by integrated pixel densitometry (A) resulted in significant reduced in vitro invasion in both oxygen conditions, the suppression was more prominent under hypoxia though and the hypoxia‐mediated induction of invasion was not detectable any longer (B).

Figure 5

ZEB1 activation in fetal stem and progenitor cells and ZEB1‐dependent motility: ZEB1‐positive cells in the neural stem cell niche of the human fetal subventricular zone (red arrowheads) as well as in lower layer of the germinal matrix (black arrow heads) (A), human fetal neural stem cells induce ZEB1 under hypoxia (B) and shRNA‐mediated ZEB1 inhibition significantly impaired their migration capacity. **P ≤ 0.0001.