Gene silencing for epidermal growth factor receptor variant III induces cell-specific cytotoxicity

Abstract

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively active mutant form of EGFR that is expressed in 40% to 50% of gliomas and several other malignancies. Here, we describe the therapeutic effects of silencing EGFRvIII on glioma cell lines in vitro and in vivo. A small interfering RNA molecule against EGFRvIII was introduced into EGFRvIII-expressing glioma cells (U87#) by electroporation resulting in complete inhibition of expression of EGFRvIII as early as 48 h post-treatment. During EGFRvIII silencing, a decrease in the proliferation and invasiveness of U87# cells was accompanied by an increase in apoptosis (P < 0.05). Notably, EGFRvIII silencing inhibited the signal transduction machinery downstream of EGFRvIII as evidenced by decreases in the activated levels of Ras and extracellular signalregulated kinase. A lentivirus capable of expressing antiEGFRvIII short hairpin RNA was also able to achieve progressive silencing of EGFRvIII in U87# cells in addition to inhibiting cell proliferation, invasiveness, and colony formation in a significant manner (P < 0.05). Silencing EGFRvIII in U87# cultures with this virus reduced the expression of factors involved in epithelial-mesenchymal transition including N-cadherin, B-catenin, Snail, Slug, and paxillin but not E-cadherin. The anti-EGFRvIII lentivirus also affected the cell cycle progression of U87# cells with a decrease in G 1 and increase in S and G 2 fractions. In an in vivo model, tumor growth was completely inhibited in severe combined immunodeficient mice (n = 10) injected s.c. with U87# cells treated with the anti-EGFRvIII lentivirus (P = 0.005). We conclude that gene specific silencing of EGFRvIII is a promising strategy for treating cancers that contain this mutated receptor. [Mol Cancer Ther 2008;7(11):3586 -97]

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes

“…A number of reports indicated paracrine secretion as paramount to glioblastoma growth, with ligands such as TGFα, IL-6, ΙL-8, HGF and heparin binding EGF playing central role, further supporting the hypothesis [16, 26, 42, 65-67]. Obviously, such result does not exclude the possibility that EGFR vIII acts in a cell intrinsic manner, as depicted by previous report on the effect of shRNA mediated silencing of EGFR vIII resulting in cell death [68]. This implies that a combination of cell-intrinsic and -extrinsic effects is relevant and so, heterogenous populations of cells are more appropriate models to study biology of EGFR vIII .…”

Inhibition of RalA signaling pathway in treatment of non-small cell lung cancer

“…In order to evaluate this hypothesis, and considering lack of specific pharmacological inhibitors for this pathway, we used gene specific silencing by small interfering RNA (siRNA) against RalA expressed from a lentiviral vector (anti-RalA virus). RNA interference (RNAi) is a physiological process that results in sequence-specific gene silencing by cleavage of the targeted messenger RNA and can be used to knock down therapeutic targets in cancer cells such as elements of pro-oncogenic signaling pathways [17,23,25]. Lentiviruses are considered to be efficient for transferring siRNA and are currently being tested in clinical trials [26][27][28].…”

“…Lentivirus particles carrying shRNA against RalA (5 -GATCCGACAGGTTTCTGTAGAAGATTCAAGAGATCTTCTACAGAAAC-CTGTCTTTTT-3 ) or scrambled shRNA (5 -GATCCTTCTCCGAACG-TGTCACGTTTCAAGA GAACGTGACACGTTCGGAGAATTTTT-3 ) (Santa Cruz Biotechnology, CA) which is not capable of silencing of any known cellular mRNA were used to infect A549 cells at MOI ∼ 10 in the presence of 5 g/ml polybrene (Santa Cruz Biotechnology, CA) in accordance to the protocol provided by manufacture and our previous work [17,23]. At the indicated times post-infection, proliferation, invasion and/or western blot assays were performed following harvesting the cells in general lysis buffer (Cell Signaling, MA).…”

Emodin Suppresses Maintenance of Stemness by Augmenting Proteosomal Degradation of Epidermal Growth Factor Receptor/Epidermal Growth Factor Receptor Variant III in Glioma Stem Cells

“…Detailed experimental procedures were described in the section ''Materials and Methods.'' *P < 0.05, **P < 0.01, ***P < 0.005. markers that are known to be involved in the invasion of glioma cell lines [30] (Fig. 4B).…”