Silencing the epidermal growth factor receptor gene with RNAi may be developed as a potential therapy for non small cell lung cancer

Abstract

Lung cancer has emerged as a leading cause of cancer death in the world. Non-small cell lung cancer (NSCLC) accounts for 75-80% of all lung cancers. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. Double stranded RNA (dsRNA)-mediated RNA interference (RNAi) has shown promise in gene silencing, the potential of which in developing new methods for the therapy of NSCLC needs to be tested. We report here RNAi induced effective silencing of the epidermal growth factor receptor (EGFR) gene, which is over expressed in NSCLC. NSCLC cell lines A549 and SPC-A1 were transfected with sequence- specific dsRNA as well as various controls. Immune fluorescent labeling and flow cytometry were used to monitor the reduction in the production of EGFR protein. Quantitative reverse-transcriptase PCR was used to detect the level of EGFR mRNA. Cell count, colony assay, scratch assay, MTT assay in vitro and tumor growth assay in athymic nude mice in vivo were used to assess the functional effects of EGFR silencing on tumor cell growth and proliferation. Our data showed transfection of NSCLC cells with dsRNA resulted in sequence specific silencing of EGFR with 71.31% and 71.78 % decreases in EGFR protein production and 37.04% and 54.92% in mRNA transcription in A549 and SPC-A1 cells respectively. The decrease in EGFR protein production caused significant growth inhibition, i.e.: reducing the total cell numbers by 85.0% and 78.3%, and colony forming numbers by 63.3% and 66.8%. These effects greatly retarded the migration of NSCLC cells by more than 80% both at 24 h and at 48 h, and enhanced chemo-sensitivity to cisplatin by four-fold in A549 cells and seven-fold in SPC-A1. Furthermore, dsRNA specific for EGFR inhibited tumor growth in vivo both in size by 75.06% and in weight by 73.08%. Our data demonstrate a new therapeutic effect of sequence specific suppression of EGFR gene expression by RNAi, enabling inhibition of tumor proliferation and growth. However, in vivo use of dsRNA for gene transfer to tumor cells would be limited because dsRNA would be quickly degraded once delivered in vivo. We thus tested a new bovine lentiviral vector and showed lentivector-mediated RNAi effects were efficient and specific. Combining RNAi with this gene delivery system may enable us to develop RNAi for silencing EGFR into an effective therapy for NSCLC.

Figure 1

SiRNA-mediated RNAi effects in A549 and SPC-A1 cells. (A) dsRNA-mediated inhibition of EGFR gene expression in A549 cells (upper) and SPC-A1 cells (lower). Fluorescence images taken 48 h after transfection were shown. (a) Cells were stained with FITC-conjugated secondary antibody. (b) Cells were stained with an EGFR-specific antibody and secondary antibody. (c) FITC staining of cells transfected with unrelated dsRNA. (d) FITC staining of cells transfected with dsRNA-EGFR. (B) EGFR gene expression was quantified in both control and transfected cells by a flow cytometry with excitation and emission settings of 488 nm and 530 nm respectively. Cells were transfected for 48 h, then stained with an EGFR-specific antibody. Results were expressed as the percentage of fluorescent intensity relative to controls. Each column represented the mean of three replicated experiments. (C) EGFR gene level was quantified by real-time PCR. Expression of EGFR mRNA was analyzed using a semiquantitative real-time PCR assay. The relative gene levels were calculated in relation to the expression of the housekeeping GAPDH gene.

Figure 2

The effects of dsRNA-EGFR on A549 (A) and SPC-A1 (B) cell count and colony formation (C). Cells were seeded into 6 well plates at a density of 1 × 10⁵ per well. Forty-eight hours after transfection, cell numbers were determined using a hemocytometer. For colony forming assay, cells transfected with siRNA,

Figure 3

Effects of siRNA-EGFR on NSCLC cells' ability to migrate. NSCLC cells in control group showed higher motility in a standard scratch assay. The migration of A549 cells (upper) and SPC-A1 cells (lower) was quantitatively assessed at time points of 24 h (white) and 48 h (black) after the introduction of a scratch in monolayer transfected cells grown on collagen IV. Each column represented the mean of three experiments; bars, SD.

Figure 4

Effect of dsRNA-EGFR on chemosensitivity of NSCLC cells to cisplatin. Cells were transfected with siRNA, then exposed to various doses of cisplatin for 48 h and viability was accessed. The percentage of cell growth was calculated by comparison of the A₄₉₀ reading from treated versus control cells. The IC₅₀ value of A549 (upper) cells to cisplatin in control group, transfection reagent control, unrelated dsRNA group and dsRNA-EGFR group was 2.67 μg/ml, 2.30 μg/ml, 2.19 μg/ml and 0.50 μg/ml respectively. The IC₅₀ value of SPC-A1 cells to cisplatin (lower) was 3.67 μg/ml, 3.11 μg/ml, 3.07 μg/ml and 0.43 μg/ml respectively.

Figure 5

Inhibition of tumor growth in vivo by dsRNA specific for EGFR. Transfected SPC-A1 cells (~1 × 10⁶) were injected into the left flank area of the mice and tumor xenografts were allowed to grow to a size of approximately 10 mm × 10 mm. After mice were sacrificed, tumors were removed and weighed. (A) Tumor volumes measured by calipers every 2 days. (B) Tumor weight. Each column represented the mean of six mice, bars, SD.