Article
Depletion of SOX2 by RNAi results in downregulation of stem cell markers and induction of amoeboid migration mode in human glioma cells
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Published: | May 20, 2009 |
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Objective: Recently, we have detected the stem cell marker SOX2 in a subset of tumor cells in glioblastoma multiforme (GBM). SOX2 represents a transcription factor which has attracted growing attention since it contributes to the maintenance of pluripotent adult neural stem cells. Its expression in GBM as well as glioblastoma cell lines suggests a role in autonomous cell proliferation and indicates a cancer stem cell-like phenotype. Since it appeared as a promising molecular target, we sought to establish an experimental siRNA therapy targeting SOX2 to treat gliomas.
Methods: In order to silence SOX2 expression in glioma cells, 9 different shRNA target sequences were designed and ligated into retroviral shRNA expression vectors. An efficient downregulation of SOX2 protein in U343 and U373 glioma cells was accomplished by transduction of shRNA molecules sh788 and sh2378, which was confirmed by Western blot and indirect immunofluorescence analysis. The RNAi-phenotype was investigated by standard proteinbiochemical analysis, confocal laser scanning microscopy and time lapse video imaging of SOX2-depleted cells.
Results: Knock down of SOX2 expression by RNAinterference in U343 and U373 glioma cells led to a cell cycle arrest in G1 and to the downregulation of stem cell markers such as Nestin and Oct4. Strikingly, SOX2-depletion also caused the GBM cells to switch to an amoeboid migration mode, characterized by actin filament remodeling and RhoA-activation. This amoeboid migration mode could be inhibited by the small chemical compound Y27632, a specific inhibitor for ROCK, a kinase downstream of RhoA.
Conclusions: We hypothesize that a switch in cellular motility, which might occur upon posttranslational modification or down-regulation of SOX2 in GBM, contributes to the invasive nature of GBM cells. Further experiments such as transcriptome analysis and biochemical approaches to identify candidate genes affected by stable SOX2-RNAi and linked to the amoeboid migration mode should elucidate the mechanism governing the amoeboid migration of glioma cells.