P11.26 Genome-wide shRNA screen identifies candidate genes driving glioblastoma invasion

Abstract

Abstract BACKGROUND A major hallmark of glioblastoma (GBM) is its highly invasive capacity, contributing to its aggressive behaviour. Since invasive cells cannot be easily removed by surgery or irradiation, they are left behind and eventually result in lethal recurrence. Therefore, a better understanding of the invasion process and of the key molecular players underlying the invasive capacities of GBM may lead to the identification of new therapeutic targets for GBM patients. MATERIAL AND METHODS To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM sphere cultures. The phenotype of the most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cell lines was compared with overexpression in non-invasive cells. RNA sequencing of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion. RESULTS Through a whole genome shRNA screen, a zinc-finger containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong decrease in invasion capacity in two highly invasive GBM cell lines. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of either one or both zinc-finger motifs led to decreased invasion indicating that the two zinc-finger motifs are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus. CONCLUSION We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through a transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent a novel promising target to overcome invasive capacities in GBM.