ted CT99021 CHIR-99021 in solid cancers were shown IC50 values were calculated based on Figures 1C and 1D. doi:10.1371/journal.pone.0026760.t001 Figure 1. Schematic representation of ERBB2 mutations analyzed. The side chains of mutants considered in this study are plotted together with a schematic representation of the protein fold using the crystal structure of EGFR kinase in complex with erlotinib. B is a view roughly orthogonal to A and shows additional inhibitors gefitinib and lapatinib superimposed at the ATP binding site. doi:10.1371/journal.pone.0026760.g001 Sensitivity of ERBB2 Mutations towards Lapatinib PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26760 with EGFR or ERBB3.
Early passage NMuMg cells stably expressing wt or mutant ERBB2 formed distinct colonies in six well cell culture plates as well as in soft agar. Hereby, ERBB2 L755S, ERBB2 L755P, ERBB2 V777L and ERBB2 T862A formed more colonies compared to wt ERBB2 indicating an enhanced transforming potential. Interestingly, late passage NMuMg cells stably BMS-536924 expressing ERBB2 L755S, ERBB2 L755P, ERBB2 V777L, ERBB2 T798M, ERBB2 T862A and ERBB2 H878Y also formed colonies in liquid culture in contrast to wt ERBB2 also supporting enhanced transforming potential of these ERBB2 mutants. Similar observations were made in a recent report with NIH3T3 cells expressing ERBB2 L755S. We next aimed to establish additional ERBB2 mutant expressing cell lines, which completely depend on the overexpressed ERBB2 for their survival.
This allows to study their sensitivity towards different kinase inhibitors in a convenient way. Thus, ERBB2 mutations were cloned into the MiGR1 vector and stable expressing Ba/F3 cell lines were established. Both wild type ERBB2 and ERBB2 mutants conferred Ba/F3 cells to cytokine independence. We then tested the inhibitory effects of lapatinib on these stable Ba/F3 cell lines expressing ERBB2 mutants. Cell proliferation analysis showed that the ERBB2 H878Y mutant had the highest sensitivity against lapatinib among all mutations tested with a cellular IC50 value nearly half to that of wild type ERBB2. A similar sensitizing effect of ERBB2 H878Y towards lapatinib was shown recently in CHO cells measuring autophosphorylation of the receptor.
Thus, ERBB2 H878Y, which was reported in 11% of hepatoma patients, can be considered as a lapatinib sensitizing mutation similar to EGFR L858R that was reported as gefitinib sensitizing mutation in NSCLC. Another mutation, ERBB2 V777L also remained sensitive to lapatinib with a cellular IC50 value similar to that of wild type ERBB2. However, all remaining mutations showed a shift towards significant higher cellular IC50 values compared to the wild type receptor. Since levels of up to 1 mM of lapatinib may be achieved in patients, ERBB2 V773A, ERBB2 T862A and ERBB2 N857S mutations might respond to higher doses of lapatinib. In contrast, ERBB2 L755S Figure 2. Biochemical analysis of ERBB2 mutants. HEK293 cells were transfected with either wild type or mutant ERBB2 for 36 hours and analyzed for autophosphorylation and activation of downstream signaling molecules. Untransfected cells were taken as control for ERBB2 expression. HEK293 cells were transfected with a combination of ERBB2 and EGFR or ERBB3 constructs for 36 hours followed by serum starvation