Effective silencing of EGFR with RNAi demonstrates non-EGFR dependent proliferation of glioma cells

Abstract

Abstract. The epidermal growth factor receptor (EGFR, ErbB1) is frequently dysregulated in a variety of solid human tumors, including malignant glioma. EGFR expression has been associated with disease progression, resistance to standard therapies and poor survival. The application of small interfering RNAs (siRNAs) has become an effective and highly specific tool to modulate gene expression, and a wide range of oncogenes have been silenced successfully. Here we show the siRNA-mediated down-regulation of EGFR in two established glioma cell lines with different EGFR expression levels (U373 MG, LN18). The expression of EGFR mRNA and protein was down-regulated by 70-90%. However, siRNA treatment had no inhibitory effect on cell proliferation, migration and activation status of EGFR-coupled signaling cascades. In accordance with these results, gene expression analysis with microarrays revealed only small, albeit specific changes in expression patterns. In conclusion, these data indicate that the specific down-regulation of EGFR might not be sufficient for a single agent therapeutic approach in malignant glioma. IntroductionMalignant glioma represent the most common primary brain tumors in the adult. Their anatomical localization, the infiltration of the surrounding normal brain parenchyma and the suppression of a tumor-directed immune response contribute to their highly aggressive phenotype. Despite extensive efforts to improve surgery, radiotherapy and chemotherapy, the median survival for patients with glioblastoma multiforme (GBM) averages only 14 months (1). One of the most frequently altered genes in glioblastoma is the epidermal growth factor receptor (EGFR, ErbB1, HER-1). EGFR gene amplification occurs in 40-50% of GBM, and tumors without amplification might also display an overexpression of this receptor (2-4). A common mutation (EGFRvIII), occurring in about half of the glioma with receptor amplification, confers enhanced tumorigenicity by rendering the receptor constitutively active (5). EGFR represents the prototype of class I receptor tyrosine kinases (RTKs). Besides EGFR, the ErbB family of receptors comprises three more members, ErbB2 (HER-2/NEU), ErbB3 (HER-3) and ErbB4 (HER-4). Receptor activation takes place after binding of specific ligands, e.g., epidermal growth factor (EGF), transforming growth factor · (TGF·), Amphiregulin, heparin-binding EGF (6). Ligand binding induces receptor homo-or heterodimerization and activation of the tyrosine kinase domain, which in turn phosphorylates both the receptor itself and downstream effector molecules. This results in signaling through multiple pathways, including the activation of extracellular regulated kinases 1/2 (ERK1/2), protein kinase B (PKB) and members of the signal transducer and activator of transcription (STAT) family. Ultimately, cells respond with enhanced proliferation, migration and transcriptional activity, as well as decreased apoptosis (7-9). Several new strategies have been developed to target EGFR, including monoclonal antibodies (mAbs) (...

Using the molecular classification of glioblastoma to inform personalized treatment

“…Hegi et al showed that the tissue drug concentrations are increased and efficient EGFR dephosphorylation takes place both in vivo and in vitro , but the downstream pathway remains active. Persistent EGFR downstream signalling has also been reported by Hasselbalch et al on glioblastoma cell lines treated with cetuximab, an anti‐EGFR monoclonal antibody, and by Vollman et al using siRNAs. These findings suggest that EGFR downstream signalling is independent of receptor activation and that simultaneous downstream signals might co‐exist or be initiated as a response to EGFR blockade.…”

Treatment of glioblastoma multiforme using a combination of small interfering RNA targeting epidermal growth factor receptor and β‐catenin

“…In agreement with previous studies , the results of the present study also revealed that down‐regulation of EGFR did not reduce cell proliferation as significantly as β‐catenin inhibition from both the cell viability and clonogenic assays, which thereby resulted in a comparable inhibitory effect of targeting β‐catenin alone versus the combinatorial treatment. There have been conflicting reports in the literature regarding the therapeutic effect of targeting EGFR against GBM.…”

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

“…siRNA-mediated knockdown of Rictor was also previously reported to have no effect on cell proliferation, survival and invasion [71]. The absence of significant therapeutic effects associated with EGFR inhibition described here also correlates with results observed in the clinic [61], [74]–[76] using small molecule EGFR inhibitors, and in some pre-clinical GBM studies [77]–[79] using small molecule EGFR inhibitors or siRNA. More specifically, Fan et al reported that the EGFR inhibitor gefitinib or the pan-PI3K inhibitor LY294002 given as monotherapy had no impact on tumor burden in a GBM model, yet when these inhibitors were used in combination, they blocked tumor growth [78].…”