RNA interference against a glioma-derived allele of EGFR induces blockade at G2M

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Abstract
Amplification and mutation of the epidermal growth factor receptor (EGFR) is common in astrocytoma. The most frequently occurring mutation (EGFR, EGFRvIII) deletes exons 2–7 from this receptor tyrosine kinase (RTK), and signals constitutively in the absence of ligand. EGFR is not found in normal tissue, and therefore represents an attractive therapeutic target. Here, we show that a small interfering RNA (siRNA) directed against the unique exon 1/exon 8 junction sequence of EGFR efficiently suppressed expression of EGFR in rodent fibroblasts and in two human glioblastoma cell lines. SiRNA-mediated depletion of EGFR led to reduction in the levels of phosphorylated Akt in glioma cells, was associated with increased apoptosis, and induced partial arrest at the G2M phase of the cell cycle. Inhibitors of PI3 kinase cooperated with siRNA treatment, leading to further increases in both cell cycle blockade and apoptosis. Importantly, cell cycle blockade could be reversed, and apoptosis rescued using a conditional allele of Akt, implicating Akt as a primary target of combination therapy. This study demonstrates the therapeutic potential of siRNA to impact EGFR as a glioma-specific target, and offers a mechanistic rationale for combining siRNA and small molecule inhibitor therapies against distinct components in the EGFR signaling pathway.