ErbB1 chains contain intracellular tyrosines some of which be come autophosphorylated by dimerization and serve as docking sites for adaptor selleck chemicals proteins that convey signals downstream thus promoting cell survival, angiogenesis, migration and tumor cell invasion. Additional phosphorylations of EGFR by other kinases stabilize and enhance receptor activity. The importance of EGFR kinase activity in lung cancer is illustrated by the approval of tyrosine kinase inhibitors as therapeutic agents. TKIs competitively bind and inhibit the catalytic kinase domain preventing EGFR from initi ating signal transduction. Targeting EGFR in lung cancer is particularly successful in patients with activation mutations in ErbB1, while other NSCLC patients either are partially responsive, have disease stabilization, or do not respond at all.

Approximately 15% of tumors in lung cancer patients exhibit EGFR activating muta tions and have significant responses to TKIs targeting EGFR. Resistant Inhibitors,Modulators,Libraries to EGFR inhibitors occurs and is associ ated with activation of additional signaling pathways, or secondary mutations in the ErbB1 gene that make EGFR less susceptible to inhibitors. Resistance and lack of responsiveness in the majority of metastatic lung cancer patients emphasize the importance of identifying additional targets for drug therapy. In some tumor cell lines, EGF Inhibitors,Modulators,Libraries receptors are activated by unknown mecha nisms, hence we reasoned that cell lines could be used to define additional proteins to target. Our approach was to delineate mechanisms of constitutive phosphoryl ation of EGFR in lung adenocarcinoma cell lines.

In preliminary studies constitutive phosphorylation of the EGFR at Y 845 and Y 992 in the Calu3 cell line was found independent of EGF stimulation. The objective of this study thus, was to determine the mechanisms lead ing to constitutive phosphorylation of EGFR. Once the mechanisms are defined, then inhibitors can be selected Inhibitors,Modulators,Libraries to Inhibitors,Modulators,Libraries counteract constitutive receptor activation. Materials and methods Cell lines tissue culture flasks, washed in PBS, then detached with Cell Dissociation Buffer. For inhibitor studies, Calu3 cells were seeded at 500,000 cells/well while H1975 cells were seeded at 750,000 cells/well and allowed to ad here overnight to achieve 80 90% confluency before serum starvation for 6 hours to overnight.

Cells were treated with various inhibitors or solvent vehicles Inhibitors,Modulators,Libraries in serum free medium as indicated. Diphtheria toxin mutant CRM197, and myristoylated PKC II peptide inhibitor I . erlotinib . U0126, and human recombinant EGF, . PP2, GM6001 and TAPI . and Enzastau rin. inhibitor Lenalidomide Erlotinib and LY317615 were obtained through Materials Transfer Agreements with OSI and Roche/ Genentech, and with Lilly Oncology, respectively. Calcein AM proliferation assay Cells were seeded at 15,000 cells per well into 96 well flat bottom plates.