Establishment of acquired resistance to epidermal growth factor receptor (EGFR) inhibitors in human tumor cell lines

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Although molecular inhibitors of EGFR signaling carry high promise in cancer therapeutics, clinical trial results to date indicate that only a small subset of patients (10-20%) achieve objective tumor responses. To investigate potential mechanisms of resistance to EGFR inhibitors, acquired resistance has been established across a panel of human tumor cell lines. Specifically, we established in vitro resistance to cetuximab (Erbitux^TM, C225), gefitinib (Iressa^TM, ZD1839) and erlotinib (Tarceva^TM, OSI-774) in human head and neck (H&N) and lung cancer cell lines. Over a period of 6 months, tumor cells in culture were continuously exposed to increasing concentrations of either anti-EGFR mAb or receptor tyrosine kinase inhibitors (TKI). Concentrations commenced at the IC50 for a particular cell line and were doubled every 10-14 days up to the maximum tolerated dose level that enabled cell viability. Once fully established, we characterized the proliferative growth profiles for resistant versus parental cells to EGFR inhibitor challenge. Following exposure to high dose gefitinib (1 uM), erlotinib (1 uM) or cetuximab (100 nM) over 16 days, the parental cell populations showed significantly greater growth inhibition than that achieved in the resistant cell populations across all time points. Across the various tumor cell lines and EGFR inhibitors tested, we observed a 10 to 30-fold increase in the resultant IC50 values to EGFR inhibitor challenge for resistant cells. Flow cytometry analysis using propidium iodide staining demonstrated that TKI- resistant H&N cells failed to demonstrate the characteristic G1 cell cycle arrest following exposure to 0.5 uM of gefitinib or erlotinib that was common for parental cells. These results correlate with the higher proliferation rates observed in EGFR-resistant cells over that observed in parental cells. Interestingly, when cetuximab-resistant cells were exposed to gefitinib or erlotinib, a dose-dependent inhibition of cellular proliferation was observed not only in parental cells but also in cetuximab-resistant cells. Conversely, when gefitinib or erlotinib-resistant cells were exposed to varying concentrations of cetuximab, consistent growth inhibition was not observed. These preliminary results suggest that cross-resistance of TKI-resistant cells to mAb-challenge has been achieved, but not cross-resistance of mAb-resistant cells to TKI challenge. Additional studies to more fully characterize differences between the EGFR-resistant versus parental cell lines are in progress, as well as studies to explore the patterns and maintenance of EGFR resistance once the cell lines are established as xenografts in athymic mice. (Cetuximab was kindly provided by ImClone Systems, gefitinib by AstraZeneca, and erlotinib by OSI/Genentech/Roche).