Elucidation of the gefitinib (‘Iressa’, ZD1839)-resistant mechanisms in acquired gefitinib-resistant human non-small-cell lung cancer cell lines
Authors: Fumiko Inoue, Tohru Ohmori, Koichi Ando, Toshimitsu Yamaoka, Masanori Matsuda, Takashi Hirose, Naoya Horichi, Kazuto Nishio, Nagahiro Saijo, Mitsuru Adachi, Carlos L. Arteaga, Toshio Kuroki
Journal: Cancer Research (2004)

1221  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’, ZD1839), an orally active EGFR-TKI (EGFR tyrosine kinase inhibitor), is clinically used in patients with non-small-cell lung cancer (NSCLC). Gefitinib is administered using a once-daily dosing regimen; however, clinical resistance may develop following long-term exposure. Some cases have already been reported, with lost sensitivity to gefitinib occurring after several months or years of administration. To elucidate a mechanism of resistance to gefitinib, we established gefitinib-resistant PC-9 NSCLC cell lines (PC-9/ZD2001, PC-9/ZD5001, and PC-9/ZD1K1) by stepwise dose escalation of gefitinib, which could survive under concentrations of 200, 500, and 1000 nM gefitinib, respectively. After more than 6 months under culture in gefitinib-free conditions, the sensitivity of PC-9/ZD2001 cells to gefitinib was restored, and subsequently established as a revertant cell line, PC-9/ZD2001R. The IC40 value (determined by MTT assay) of gefitinib in PC-9 cells was approximately 52.7 nM. The gefitinib-resistant cell lines showed a 4 to 27-fold higher resistance to gefitinib (IC40 =211, 841, 1400 nM) than PC-9 cells. The sensitivity to gefitinib was completely restored in PC-9/ZD2001R cells. Interestingly, the gefitinib-resistant cell lines were about 3-fold more sensitive than PC-9 cells to the cytotoxic effects of paclitaxel, vincristine, and vinorelbine. These collateral sensitivities were diminished in PC-9/ZD2001R cells. In all of the resistant cell lines, expression levels of EGFR and mRNA decreased to 10-30 % of that in parental cells. There were no differences in EGFR DNA-sequences or autophosphorylation levels of specific phospho-tyrosine sites (tyr845, tyr992, tyr1045, tyr1068), between PC-9 and the resistant cell lines. EGFR signaling was dissociated downstream; the inhibitory effect of gefitinib on mitogen-activated protein kinase (MAPK) and Akt pathways were significantly decreased in resistant cells. Similar dissociation was obtained in PC-14, an intrinsic gefitinib-resistant NSCLC cell line. In PC-9/ZD2001R cells, the decrease in EGFR and mRNA expression was almost the same as in the PC-9/ZD2001 cells, however, the association between EGFR and MAPK signaling was completely reconstituted. These results suggest that a decrease in EGFR expression and dissociation of EGFR from the MAPK signaling pathway should contribute to acquired gefitinib-resistance. The dissociation mechanisms, which include expression levels of EGFR/MAPK related molecules in gefitinib-resistant cells, should be further investigated. ‘Iressa’ is a trademark of the AstraZeneca group of companies