Artikel
Hypoxia-mediated induction of EMT activators increases in vitro invasion of glioblastoma-derived cell cultures
Suche in Medline nach
Autoren
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Ulf D. Kahlert
- Department of Pathology, Division of Neuropathology, Johns Hopkins Hospital, Baltimore, MD, USA
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Florian A. Siebzehnrubl
- Department of Neurosurgery, University of Florida, Gainesville, FL, USA
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Eli E. Bar
- Department of Neurosurgery, School of Medicine, Case Western Reserve University, Cleveland, OH, USA
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Charles G. Eberhart
- Department of Pathology, Division of Neuropathology, Johns Hopkins Hospital, Baltimore, MD, USA
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Jaroslaw Maciaczyk
- Abteilung Funktionelle und Stereotaktische Neurochirurgie, Universitätsklinikum Düsseldorf
| Veröffentlicht: | 13. Mai 2014 |
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Gliederung
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Objective: Hypoxia is thought to induce migration in many neoplasms, often by promoting epithelial-to-mesenchymal transition (EMT), but the mechanisms by which it affects brain tumor invasion is less well understood. In particular, little is known about the possible role of EMT-associated factors in hypoxia-mediated induction of migratory phenotype in gliomas.
Method: Migration and invasion of GBM-derived cell lines (n5) cultivated as spheres under atmospheric oxygen (21% O2) and acute (14h) or chronic (48h) hypoxia (1% O2) have been assessed in Boyden Chamber-based in vitro assay. Digoxin has been used to inhibit the HIF-1-mediated hypoxic response. ZEB1 knockdown has been achieved using lentiviral sh-RNA delivery. The quantitative gene expression and protein analyses have been performed according to standard real-time qPCR or Western Blot protocols respectively. Standard immunohistochemistry on formalin-embedded GBM-patient tumor samples to detect ZEB1-positve cells has been applied. Prognostic value of EMT-associated factors in a cohort of GBM patients has been assessed based on publically available genomic and clinical databases (TCGA and Rembrandt).
Results: Hypoxia led to a significant increase of invasion (up to 6 fold) in all tested cell lines, as assessed by Boyden Chamber-based assay. This was accompanied by an induction of EMT activators including ZEB1, SNAI1 and TWIST1 (up to 8 fold) as compared to normoxic cultures. Digoxin treatment resulted in clear abolition of this phenomenon. Moreover, ZEB1-knockdown has significantly reduced the invasive behavior of the cells. The histological analysis of patient-derived GBM samples revealed an abundance of ZEB1 positive cells in hypoxic, peri-necrotic tumor areas. Furthermore, the up-regulation of EMT activators such as TWIST1, SNAI1 and SNAI2 was found as a hallmark of the most aggressive, mesenchymal subgroup of GMBs in the analysis of TCGA and Rembrandt datasets.
Conclusions: Hypoxia increases the invasion capacity of GBM-derived cells inducing mesenchymal developmental program by the process of epithelial-to-mesenchymal-like transition. Inhibition of the hypoxic, HIF-1-mediated molecular changes protects from over-expression of EMT activators and might contribute to the development of the novel therapeutic strategies aiming at the elimination of the migratory cell subpopulation, and thus preventing the tumor dispersion.
