Figure 1

Lapatinib's molecular functioning involves AMPK-dependent release of extracellular fatty acid synthase (FASN). (a) HER1 or HER2 inhibitors (or both) differentially regulate extracellular expression of FASN. FASN concentration in cell culture supernatants from SKBR3 cells cultured in low-serum (0.5% fetal bovine serum)-containing Dulbecco's modified Eagle's medium in the presence or absence of cetuximab (100 μg/mL), trastuzumab (100 μg/mL), erlotinib (1 μmol/L), gefitinib (1 μmol/L), or lapatinib (1 μmol/L) for 48 hours was measured by enzyme-linked immunosorbent assay (ELISA) as described elsewhere [2]. (b) HER1 or HER2 inhibitors (or both) differentially regulate the activation status of AMPK. After the harvesting, cell cultures treated as described above were lysed and prepared for immunoblotting as described elsewhere [2]. A representative immunoblotting analysis obtained by using the AMPK Antibody Kit (Cell Signaling Technology, Inc., Danvers, MA, USA) is shown. (c) Lapatinib treatment induces extracellular release of FASN in an AMPK-dependent manner. SKBR3 cells were transiently transfected with AMPK short interfering RNA (siRNA) or control siRNA or were treated with transfection reagent alone (mock) before exposure to 1 mmol/L lapatinib for 48 hours. The FASN-detect ELISA was then used to quantitatively assess the released concentration of FASN in the extracellular milieu [2]. (d) Lapatinib-induced release of extracellular FASN is related to lapatinib efficacy in trastuzumab-refractory breast cancer cells. (e) Lapatinib-induced release of extracellular FASN is related to lapatinib's molecular functioning and involves activation of AMPK. Results in graphs are means (columns) and 95% confidence intervals (bars) of two independent experiments made in triplicate. Statistically significant differences (one-factor analysis of variance [ANOVA]) between experimental conditions and unsupplemented control cells are shown by asterisks (*P < 0.01, **P < 0.001; n.s., not statistically significant). All statistical tests were two-sided. AMPK, AMP-activated protein kinase.