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EGFR-targeted therapy-induced resistance mechanism in glioblastoma

ABSTRACT: Analysis of mouse glioma tumors (in RAG1-/- mouse host) overexpressing Control(C) or Slug(S) viruses, and treated with doxycyclin(D) to turn off EGFRviii expression. Results provide insight into molecular basis of EGFRviii targeted therapy-induced resistence in GBM. By utilizing animals engineered with doxycycline (dox)-off oncogenic EGFRvIII transgene and conditional Ink4a/Arf and Pten alleles (Nestin-CreERT2; Ink4aL/L; PtenL/L; hGFAP_tTA; tetO-EGFRvIII, designated iEIP), we previously demonstrated that sustained oncogenic EGFR signaling was required for maintenance of EGFR-driven glioma progression and suppression of oncogenic EGFRvIII transgene expression induces tumor regression. But despite of a robust initial response, the regressed tumors relapsed inevitably. The finding that relapsed tumors had escaped oncogenic EGFR signaling addiction promoted our search for potential genetic events that might fuel the resistance development. Three relapse and two matched treatment-naïve tumors derived from the same parental line were analyzed by whole exome sequencing.

ORGANISM(S): Mus musculus

PROVIDER: GSE108658 | GEO | 2019/01/01

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.

Project description:Background: EGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2-7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation. Methods: We constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis,quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis. Results: In the course of screening 30,000 substances, a reagent, âErtredin,â was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed both oxidative phosphorylation and glycolysis under 3D conditions, and promoted cell apoptosis. Conclusion: We developed a high throughput screening method based on anchorage-independent sphere formation induced by EGFRvIII-dependent transformation. In the course of screening, we identified Ertredin, which inhibited anchorage-independent 3D growth and tumor formation in nude mice. Functional analysis suggests that Ertredin suppresses both mitochondrial oxidative phosphorylation and cytosolic glycolysis in addition to promoting EGFRvIII degradation, and stimulates apoptosis in sphere-forming, EGFRvIII-overexpressing cells. Patterns of gene expression in cells treated with Ertredin (sample 2), rotenone (sample 3), and AG1478 (sample 4) were compared with vehicle-control cells (sample 1).

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EGFR-targeted therapy-induced resistance mechanism in glioblastoma

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