Abstract
Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.
Highlights
Glioblastoma is the most common malignant primary brain tumor in adults and carries a poor prognosis with limited therapeutic options
We generated a stable U138 cell line expressing Cas[9] fused to blue florescent protein (BFP). These transduced cells were subsequently infected with lentiviral sgRNA libraries targeting 4,574 genes which were predicted to be involved with cell motility or serve as drug targets (sub-libraries used (1) drug targets, kinases phosphatases and (2) trafficking, mitochondrial, motility)[12]
This population included expected hits such as PDGFRα, which has previously been shown to be necessary for glioblastoma invasion, suggesting our assay was capable of identifying known regulators of migration[14,15]
Summary
Glioblastoma is the most common malignant primary brain tumor in adults and carries a poor prognosis with limited therapeutic options. To establish the importance of MAP4K4 function in cell motility for glioblastoma beyond the U138 human glioma line used for this screen, we used a specific drug inhibitor to demonstrate reduced migration in a wound healing assay in three additional adult malignant glioma lines. Using this drug in a human tumor organotypic slice culture limited migration in a subset of gliomas. We used CRISPR-Cas[9] to screen for genes essential for human glioblastoma invasion and found that MAP4K4 plays an important role in brain tumor invasion.
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