Enhancement of Epidermal Growth Factor Receptor-degradation Pathway in Acquired Gefitinib-resistant Human Non-small Cell Lung Cancer Cell Lines

The epidermal growth factor receptor (EGFR) is highly expressed in a range of tumor types, and has been implicated in several aspects of tumor survival and growth, such as cell proliferation, apoptosis, angiogenesis, and metastasis. Gefitinib (‘Iressa’, ZD 1839), an orally active EGFR-TKI (EGFR tyrosine kinase inhibitor), is clinically used in patients with non-small-cell lung cancer (NSCLC) . Because this drug is continuously administered until recurrence of the disease or treatment is stopped for other reasons, clinical drug resistance might develop during long-term exposure. To elucidate the mechanism (s) of resistance to gefitinib, we established gefitinib-resistant NSCLC cell lines using a stepwise-dose escalation method. This resistant cell line, PC-9/ZD201 had a 3- to 4-fold increase in resistance to gefitinib compared to parental PC-9 cells. After more than 6 months of culturing in drug-free conditions, PC-9/ZD201 regained sensitivity to gefitinib, this revertant cell line was named PC-9/ZD201 R. In PC-9/ZD201 cells, EGFR mRNA and protein expression levels decreased to less than 50% of those in PC-9 cells. In PC-9/ZD201R cells, EGFR protein but not mRNA expression partially recovered. After 2 hrs exposure to 100 ng/ml Transforming Growth factor α (TGF-α ), EGFR protein expression was significantly decreased in resistant cells but not in the parental PC-9 cell line. This decrease in EGFR expression was completely inhibited by lactacystin, a 26S proteasome inhibitor. To determine EGFR-degradation activity in gefitinib-resistant cell lines, EGFR-internalization assays and -degradation assays were performed using 125I-EGF. EGFR-internalization and -degradation activities were increased about 2 fold in PC-9/ZD201 compared to PC-9 and PC-9/ZD201R cell lines. In intrinsic peftinib-resistant PC-14 cells, EGFR-degradation was more than 5-fold higher than in PC-9 cells. These results suggest that the EGFR-ubiquitylation and -degradation pathways contribute to the development of gefitinib-intrinsic, and -acquired resistance in NSCLC cells.