499. Inhibition of Non-Small Cell Lung Cancer by Silencing EGFR with RNAi

Abstract

Top of pageAbstract Lung cancer is a leading cause of cancer death in the world. NSCLC accounts for 75–80% of all lung cancers and has a low five-year survival rate. Current therapies are unsatisfactory. RNAi has shown enormous promise in gene silencing, the potential of which in developing new methods for cancer therapy is tested in this study. We report RNAi induced silencing of EGFR gene, which is overexpressed in NSCLC. A NSCLC cell line, SPC-A1 was transfected with EGFR sequence- specific dsRNA including non-specific controls. We demonstrated a significant, sequence specific silencing of the EGFR with reduction in EGFR protein production and decrease in the number of the EGFR in SPC-A1 cells by the transfer of the dsRNA-EGFR, but not by controls. The number of the EGFR assessed by a flow cytometry was also in agreement in which dsRNA-EGFR dramatically reduced EGFR gene expression to levels that were 71.3% less than those seen in control groups. Further molecular analyses revealed that the amounts of EGFR mRNA were strongly downregulated in cells treated with dsRNA-EGFR by 54.9% while only slightly inhibited in control groups. To investigate the functional effect of the the EGFR gene silencing, we performed a cell count and a cell colony assay. Cell count results showed that the numbers of cells in the mock control, empty vector group, and unrelated dsRNA transfected groups are (33 ± 5) ×104, (32 ± 4) 104, and (28 ± 3) ×104 respectively, while in the dsRNA-EGFR group, the cell numbers were only (15 ± 2) ×104. This showed a significant decrease in the numbers of cells by 78.3% (P<0.001). The results of the colony assay were similar. The control groups showed little decrease in colony formation. However, dsRNA-EGFR group showed a significant decrease by 66.8%. These results demonstrated dramatic growth inhibition of tumor cells after transfection with dsRNA-EGFR. The EGFR silencing also retarded the migration of SPC-A1 by more than 80% both at 24 h and at 48 h, enhanced the chemosensitivity to cisplatin by seven-fold. Furthermore, dsRNA-EGFR transfection also significantly delayed tumor growth in vivo both in size reduction by 75.06% and in weight reduction by 73.08% when NSCLC cells transfected with dsRNA-EGFR were implanted. To improve gene delivery to NSCLC cells in vivo, we constructed a lentiviral vector based on the Jembrana disease virus (JDV) that contains a polymerase-III H1- RNA promoter. This promoter directs transcription of RNA with well-defined start site and a termination signal, resembling the end of synthetic siRNA. A 56 bp DNA insertion that contains 19 bp of the EGFP in sense and anti-sense orientation were cloned and packaged into VSV-G pseudotyped JDV vectors. Gene transfers to SPC-A1 and A549 with these JDV vectors were at an efficiency of 75–80% and RNAi-mediated gene silencing of the EGFP was between 75–80%. These results indicated a blockbuster effect of RNAi-mediated EGFR suppression, causing inhibition of cancer cell proliferation and motility, enhancement of chemosensitivity and impairment of tumor growth in vitro and in vivo. JDV vector may abolish the cell line differences in gene transfer. Taken together, we believe it is possible to develop the RNAi approach into an effective therapy for NSCLC.