for 12 hours. Cells and analyzed for the activation of ERBB2 as well as downstream signaling pathways by western blotting. Untransfected cells were used as control. doi:10.1371/journal.pone.0026760.g002 Sensitivity of ERBB2 Mutations towards Lapatinib PLoS ONE Tosedostat CHR2797 | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26760, ERBB2 L755P and ERBB2 T798M caused strong lapatinib resistance. These results indicate that the amino acids L755 and T798 in ERBB2 are critical residues determining lapatinib sensitivity and those patients with these mutations may not respond to lapatinib treatment. In summary, based on lapatinib sensitivity, ERBB2 kinase domain mutations can be classified into three groups: lapatinib sensitizing ERBB2 H878Y & ERBB2 V777L, lapatinib sensitive ERBB2 V773A, ERBB2 N857S & ERBB2 T862A and lapatinib resistant ERBB2 L755S, ERBB2 L755P & ERBB2 T798M.
Breast cancer patients with wild type ERBB2 kinase may develop secondary BMS 777607 resistance to lapatinib due to kinase domain mutations similar to secondary drug resistance reported in NSCLC or CML patients treated with kinase inhibitors. To test the hypothesis whether ERBB2 resistance mutations identified above can lead to secondary drug resistance in vitro we performed a classical drug resistance screen as described before using 2 mMof lapatinib. Indeed we were able to recover secondary resistance mutations in this screen indicating the possible emergence of resistance mutations in WT ERBB2 patients treated with lapatinib.
Interestingly, ERBB2 L755S was also reported recently in an in vitro lapatinib resistance screen performed at concentrations 0.4 mM, 0.6 mM, 0.8 mM and 1.2 mM. Thus, comprehensive sequence analysis of secondary lapatinib resistant patients will be necessary in the future to determine whether this is a clinically important resistance mechanism in breast cancer patients as already demonstrated in CML or NSCLC patients. We next tested whether ERBB2 kinase domain mutations exhibit differential sensitivity towards an alternative reversible ERBB2 inhibitor, AEE788. Interestingly, overall the efficacy of this inhibitor was not altered by most mutations except ERBB2 L755S, ERBB2 L755P and ERBB2 T798M. While ERBB2 L755S and ERBB2 L755P mutants remained sensitive to AEE788 at very high concentrations, the gatekeeper ERBB2 T798M mutation is totally resistant to AEE788 treatment.
Thus, lapatinib and AEE788 indeed display differential sensitivities to most ERBB2 mutants while ERBB2 L755S, ERBB2 L755P and ERBB2 T798M showed cross resistance to both inhibitors. Figure 3. Anchorage independent growth of ERBB2 mutants. NMuMg cells stably expressing either wild type or mutant ERBB2 were tested for their transforming ability in a soft agar assay. NMuMg cells infected with empty vector were used as control. 2.56104 cells/well were plated in a six well plate and analyzed after 4 weeks. Average number of colonies for each cell line was shown as compared to vector transfected NMuMg cells. doi:10.1371/journal.pone.0026760.g003 Sensitivity of ERBB2 Mutations towards Lapatinib PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26760 Structural basis of lapatinib resistance Structural modeling was performed to elucidate the possible mechanisms for lapatinib resistance due to ERBB2 kinase domain mutations. To da