shRNA hairpin sequences are supplied in the Supplemental Materials and Practices. Individual EGFR and HER2 cDNA development regions were cloned in to the pENTR/D TOPO conjugating enzyme vector and mutants were constructed with Quick-change Site Directed Mutagenesis Kit based on the manufacturers guidelines. All constructs were confirmed by DNA sequencing. Constructs were cloned in to the plenti IRES GFP lentiviral vector and infections were done as described previously. Stream Cytometry BT 474 cells were transfected with ERBB3 siRNA for 48hrs, then treated with AZD6244 or GDC 0941 for 72hrs. As described previously cells were obtained and stained with propidium iodide and AnnexinV. Cells were examined utilizing a BD LSR3 logical flow cytometer. Apoptosis was determined using the sum of AnnexinV PI/AnnexinV and positive double positive cells. Tandem mass spectrometry EGFR or HER2 was immunoprecipitated Metastatic carcinoma from cells treated with AZD6244 using anti EGFR antibody or an anti HER2 antibody, separated by SDS/PAGE, stained with Coomassie blue. Samples were prepared and groups were excised and reviewed by reversedphase microcapillary/tandem mass spectrometry as described previously and further detailed in the Supplemental Materials and Methods. RESULTS MEK inhibition contributes to activation of ERBB3/PI3K/AKT We formerly observed that AKT phosphorylation increased in reaction to MEK inhibition in HER2 amplified and EGFR mutant cancer cells. We treated HER2 amplified or EGFR mutant cell lines with the very selective allosteric MEK1/2 chemical, AZD6244, to ascertain whether this potential feedback is noticed in numerous EGFR or HER2 addicted cancer models. This MEK chemical was used at a focus of 2uM, which enough inhibited ERK1/2 phosphorylation within the HCC827 cell line. Similar effects were observed using two distinct allosteric MEK inhibitors, GSK212 and PD0325901. In each cell buy Lapatinib line, we noticed increased AKT phosphorylation at both S473 and T308 following AZD6244 treatment, as well as increased phosphorylation of several AKT targets including PRAS40, ATP citrate lyase, and GSK3/B. We established these proteins were AKT substrates, as cotreatment with an allosteric AKT inhibitor blocked their phosphorylation. MEK inhibition also resulted in up regulation of phospho CRAF and phospho MEK, suggesting activation of the popular upstream signaling molecule. This feedback also occurred in vivo, as we observed increased phospho AKT in a EGFR mutant H1975 xenograft model treated with AZD6244. Increased AKT phosphorylation suggested a potential increase in the abundance of PIP3. Consequently, EGFR driven HCC827 and HER2 driven MDA MB 453 cells were treated with a MEK inhibitor, lipids were isolated, and PIP3 levels were quantified. In both cell lines, AZD6244 induced considerable increases in PIP3.