Figure 2.

PIK3CA mRNA expression (A) and PI3K activity (B) in lung cancer cell lines. A, PIK3CA mRNA expression was compared among cell lines having different features, such as PIK3CA alterations or mutations of other genes involved in the EGFR signaling pathway. PIK3CA mRNA expression was expressed as a relative value compared with the mean of values of six HBEC cell lines. PIK3CA mRNA expression in PIK3CA gain or EGFR mutant cell lines was significantly increased compared with that of wild-type cell lines. However, PIK3CA mutant lines do not express increased mRNA levels. Horizontal bars indicate mean values. The Kruskal-Wallis test with Dunn's multiple comparison test was used to determine significance. B, equal amounts of protein (500 μg) were immunoprecipitated with anti-PI3K p85 antibody. The PI3K activity of each precipitate was then measured by ELISA and expressed as the relative production of phosphatidylinositol 3,4,5-triphosphate (PIP₃) in each sample using a commercially available ELISA kit. PIP₃ produced by NCI-H1299 cells (being wild-type for EGFR, KRAS, HER2, BRAF or PIK3CA, PIK3CA copy number 2.45) was set as 100 (control), and others were compared with this value. Each assay was done in triplicate. Mean values of PI3K activity in PIK3CA mutant cell lines were significantly higher than those of wild-type cell lines. Two cell lines having mutations of EGFR or HER2 also had relatively high PI3K activity, indicating that mutant HER2 or EGFR activates PI3K, which is downstream of these genes. Error bars indicate SD. The Mann-Whitney U test was used to determine significance.