PIK3CA mutation uncouples phosphoinositide 3-kinase signaling from HER2 inhibition by lapatinib. (A) BT474 and SKBR3 cells infected with wild-type, E545K or H1047R constructs were treated with lapatinib at the indicated doses, and lysates were analyzed by immunoblotting with the indicated antibodies. (B) Lysates from PIK3CA wild-type or mutant expressing cells treated with a range of lapatinib doses (0.0016 to 5 μM) were analyzed by ELISA for pHER2, pAkt and pS6. Half-maximal concentration (IC50) values were calculated, and the mean log IC50 ± SEM values for three replicate dose–inhibitor curves are shown. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. (C) HER2+ cell lines with wild-type PIK3CA (BT474 or SKBR3) or with a PIK3CA mutation (MDA361, HCC1954, SUM190 or UACC893) were treated with varying lapatinib doses and analyzed as described in (B). Mean log IC50 values from three replicates ± SEM are shown. Mean IC50 data are shown in Table 1.