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A genome-wide screen identifies E2F6 as critical factor for TMZ chemoresistance in EGFRvIII expressing GBM (CRISPR library RNA-seq)

ABSTRACT: We conducted a genome-wide screen by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 library to identify genes which are particularly resistant to EGFRvIII GBM cells under the chemo-stress of TMZ. In vitro and in vivo validation of EGFRvIII-specific targets revealed several strong hits, including E2F6. Mechanistic studies demonstrated that E2F6 reveals its resistance to TMZ through NF-κB and TMZ inducement.EGFRvIII GBM cells under the chemo-stress of TMZ.

ORGANISM(S): Homo sapiens

PROVIDER: GSE112734 | GEO | 2020/06/04

REPOSITORIES: GEO

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Project description:Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvoiront, this is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using a series of isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. Thus, we observed that EGFRvIII expression alters several properties of glioma EVs, including their output and global protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRVIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing cells, and down-regulation of exosomal markers (CD81 and CD82) in EVs of their indolent counterparts. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of EVs contained specific proteins such as EGFR, CD9, or others. Notably, cells expressing EGFRvIII released ample EVs double positive for CD44/BSG, and these proteins also co-localized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for development of EV-based cancer biomarkers.

Dataset Information

A genome-wide screen identifies E2F6 as critical factor for TMZ chemoresistance in EGFRvIII expressing GBM (CRISPR library RNA-seq)

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