2002;99:2866C2871

2002;99:2866C2871. described (11). The PCR primers and conditions are available upon request. siRNA and Transient Transfections HCC827 and BT-474 cells were transfected with 50nM silencer select validated siRNA or negative control (Ambion) with HiPerFect Transfection Reagent (Qiagen) according to manufacturers instructions. Transient transfections of CHO-KI cells were performed with wild-type and mutant cancers (19), suggesting Nedd4l that cancers not driven by EGFR or HER2 may have alternate, ERBB3-independent, mechanisms of MEK-inhibitor induced feedback activation of AKT. Our data suggest that the effect of MEK inhibition on ERBB3 is a novel feedback mechanism, distinct from mTORC1 feedback on IGF-IR/IRS-1. A model describing these findings is shown in Figure 4C. MEK inhibition results in increased tyrosine phosphorylation of ERBB3 due to inhibition of ERK-mediated threonine phosphorylation of EGFR and HER2 We investigated the mechanism leading to increased ERBB3 phosphorylation following MEK inhibition. HRG ligand expression was not increased with AZD6244 (Supplemental Figure 6); however, MEK inhibitor-induced feedback activation of AKT required EGFR or HER2 kinase activity (Supplemental Figure 7). Indeed, even in EGFR or HER2. 48hrs post transfection cells were treated with AZD6244 (2M) for 90 minutes. Cell lysates were immunoblotted to detect indicated proteins. Cells expressing EGFR T669A were also treated with 50ng/mL HRG ligand for 30 minutes to achieve maximal ERBB3 phsophorylation. (C) HCC827 cells were infected with a control or shEGFR hairpin, followed by infection with lentiviral vectors expressing GFP, T669 wild-type EGFR (exon 19del), or EGFR T669A (exon 19del). Following knockdown and puro selection for 72hrs, cells were treated with AZD6244 (2M) for 6hrs. Cell lysates were immunoblotted to detect the indicated proteins. Open in a separate window Figure 7 Model of MEK inhibitor-induced feedback on ERBB receptor signaling pathwaysIn untreated cells EGFR is phosphorylated at T669 by MEK/ERK, which inhibits activation of EGFR and ERBB3. In the presence of AZD6244, ERK is inhibited and T669 phosphorylation is blocked, increasing EGFR and ERBB3 tyrosine phosphorylation and up-regulating downstream signaling. To determine if this feedback model explains the activation of PI3K signaling in EGFR-mutant cancers, Nemorubicin we used shRNA to knockdown endogenous EGFR (which carries an exon 19 deletion) in the HCC827 NSCLC cell line and replaced with either EGFR (exon 19del) wild-type at T669, or EGFR (exon 19del) carrying a T669A mutation. Of note, this is the Nemorubicin same EGFR-mutant cell line in which we observed that EGFR T669 is phosphorylated in MEK-dependent manner (Figure 5, Supplemental Figure 8A). When endogenous EGFR was replaced with EGFR (exon19del) wild-type at T669, MEK inhibition led to significant feedback activation of ERBB3/PI3K/AKT signaling (Figure 6C). However, replacement with the EGFR (exon19 del) T669A mutant led to increased tyrosine phosphorylation of both EGFR and ERBB3, and activation of PI3K/AKT signaling, mimicking the effect of MEK inhibition (Figure 6C). As expected, addition of AZD6244 failed to further augment ERBB3 and AKT phosphorylation in cells expressing the 669A mutant. These results demonstrate that EGFR T669 phosphorylation is necessary for MEK/ERK to suppress EGFR-mediated Nemorubicin activation of ERBB3. This supports the hypothesis that a dominant ERK feedback on ERBB3/PI3K/AKT is mediated though phosphorylation of T669 on EGFR (or T677 HER2). DISCUSSION RAF and MEK inhibitors are being developed as treatments for cancers with.