epidermal growth factor receptor (EGFR) is a central regulator of proliferation and progression in human cancers. and subsequent EGFR-dependent activation of HER3. Taken together these findings suggest a rationale for the clinical evaluation of combinatorial anti-HER targeting approaches in tumors manifesting acquired resistance to cetuximab. following long-term exposure to cetuximab in NSCLC (H226) and HNSCC (SCC-1) cell lines. Following establishment of stable clones we performed high-throughput screening to examine the activity of 42 membrane receptor tyrosine kinases (RTKs). Through comparative analysis of cetuximab-resistant versus parental lines we identified Torin 1 that EGFR along with HER2 HER3 and cMET are all highly activated in the resistant clones. Further studies suggest that acquired resistance to cetuximab displays dysregulation of EGFR internalization/degradation and subsequent EGFR-dependent activation of HER3. RESULTS Establishment of cetuximab-resistant lines We founded cetuximab resistant tumor cell lines using the human being NSCLC collection NCI-H226 (H226) and the HNSCC collection UMSCC-1 (SCC1) to use as a model system to elucidate molecular mechanisms of acquired-resistance to cetuximab. These lines were chosen based on three main criteria; 1) Cetuximab is used in therapy for both tumor types 2 the cell lines are sensitive to cetuximab and 3) the cell lines have no TKD mutations. To generate resistant lines H226 and SCC1 cells were continually exposed to increasing concentrations of cetuximab over six months. Following the development of heterogeneous populations of cetuximab-resistant cells we isolated individual subclones GRK5 of cetuximab-resistant lines. This process resulted in six stable resistant clones for the H226 NSCLC collection designated HC1 HC4 HC5 HC6 HC7 and HC8. The sensitive parental collection was designated HP. For the SCC1 HNSCC collection six stable resistant clones were generated (SC1 SC2 SC5 SC6 SC7 SC8). As demonstrated in Number 1A all HC clones displayed a strong cetuximab-resistant phenotype when challenged with increasing concentrations of cetuximab as compared to parental controls. Related results were observed with the SCC1 cetuximab-resistant clones (Number 1B). Sequence analysis of the EGFR TKD in H226 cells after the establishment of resistant clones indicated no mutations developed during the selection process in either the resistant or parental cells (data not shown). Number 1 phospho-receptor tyrosine kinase (RTK) array in NSCLC H226 and HNSCC SCC1 cells demonstrate upregulation of EGFR HER2 HER3 and cMET Upregulation of EGFR and activation of HER2 HER3 and cMet After successful establishment of Torin 1 cetuximab-resistant clones we performed high-throughput comparative analyses measuring phosphorylated RTKs in the resistant vs. parental lines to test the hypothesis that acquired resistance to EGFR inhibition results from the activation of option RTKs that share overlapping transmission transduction elements with the EGFR. To test this hypothesis we screened the activity of a panel of triggered RTKs using an antibody-based array from R&D Systems (Minneapolis MN) as demonstrated Torin 1 in Number 1C. Following quantification of scanned images using ImageQuant software the relative manifestation of specific phosphorylated RTKs between cetuximab-resistant Torin 1 and parental cells was identified (Number 1D). The identical experimental approach was performed using the SCC1 cetuximab-resistant lines and parental control (Number 1E and F). From this high-throughput display several phosphorylated RTKs were notably up-regulated in..