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BACKGROUND: Glioblastoma (GBM) frequently displays amplification and/or mutation of the epidermal growth factor receptor (EGFR) gene. Highlighting the importance of EGFR in the pathogenesis of GBM, aberrant EGFR (ΔEGFR, also known as EGFRvIII) confers a variety of biological effects upon its expression, including resistance to radiation and chemotherapeutic agents, promotion of tumor cell motility and invasion, enhancement of tumorigenicity in vivo, and maintenance of GBM growth and heterogeneity. We hypothesized that this diverse oncogenic pathophysiology exerted by ΔEGFR is regulated, in part, through the modulation of microRNA (miR) activity, widely shown to be involved in many biological processes including cancer initiation, maintenance and progression. METHODS: To test this hypothesis, we used miR microarrays to compare miR profiles of GBM cells with activated wild type EGFR (wtEGFR) and ΔEGFR, to cells with non-activated wtEGFR or kinase-dead ΔEGFR. miRs demonstrating differential expression were validated by northern blot analysis and qRT-PCR and potential pathways regulating miR expression were interrogated by pharmacological and genetic analysis. Candidate miR targets were validated by luciferase reporter assays and western blot analysis. The role of validated miRs and targets, in modulating ΔEGFR-mediated tumorgenicity, was analyzed by overexpression and knockdown studies in engineered glioma cell lines engrafted in athymic nude mice. Validated target significance was determined by survival-association expression analysis in a patient cohort of 67 newly diagnosed and 64 recurrent GBM cases. RESULTS: We observed and validated that ΔEGFR suppresses miR-9. The repression of miR-9 was due to the negative regulation of the primary miR-9 encoding transcript, pri-miR-9-2, by ΔEGFR. Downstream of ΔEGFR, the Ras/PI3K/AKT axis was required to suppress miR-9. We identified the transcription factor, FOXP1, to be a bona-fide miR-9 target. Upregulation of miR-9 decreased expression of FOXP1 in ΔEGFR cells, suggesting that miR-9 and FOXP1 may regulate, in part, ΔEGFR-dependent GBM growth. Consistent with this, miR-9 antagonized the tumor growth advantage conferred by ΔEGFR while FOXP1 knock-down inhibited the growth of ΔEGFR-driven tumors. Upregulation of FOXP1, as a consequence of inhibiting miR-9 activity, increased the tumorigenicity of GBM cells, suggesting that miR-9 is a tumor suppressor while FOXP1 likely functions as an oncogenic factor in GBM. Finally, high FOXP1 expression was significantly associated with poor survival in GBM patients, further supporting the notion that FOXP1 is an oncogenic factor. CONCLUSIONS: Collectively, these data reveal a novel regulatory mechanism by which ΔEGFR signaling through the Ras-PI3K-Akt pathway suppresses miR-9, resulting in FOXP1 expression and increased tumorigenicity. SECONDARY CATEGORY: n/a.

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