3790

Background: Despite reproducible activity of the epidermal growth factor receptor (EGFR) inhibitors as single agents in patients with squamous cell carcinoma of the head and neck (SCCHN), the majority of patients will not have objective responses although there is nearly universal expression of the wild-type receptor. The mechanisms of this intrinsic resistance are unknown. We hypothesized that sensitivity to EGFR inhibitors can be predicted based on the inhibitors’ effects on downstream signaling. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromid (MTT) viability assays were used to assess sensitivity to the EGFR inhibitor gefitinib in 12 SCCHN cell lines. Inhibition of several downstream signaling proteins was assessed by Western blotting. The efficacy of gefitinib in combination with inhibitors of Janus kinase (JAK), STAT3, Src kinase, AKT, and phosphatidylinositol 3-kinase (PI3K) was assessed by the MTT assay. Fluorescence in situ hybridization (FISH) was performed to detect gene amplification of EGFR. Results: Based on IC50s obtained from MTT assays, cell lines were classified as sensitive, intermediate, or resistant. FISH showed that only two of the sensitive lines, SQ-20B and HN-5, were gene-amplified for EGFR. Western blotting confirmed that phosphoEGFR was inhibited at low concentrations of gefitinib in all lines tested. AKT phosphorylation, however, was inhibited only in sensitive but not resistant lines. The inhibition of STAT3 phosphorylation was observed at low concentrations of gefitinib in sensitive lines but inhibition of phosphoSTAT3 was not as consistent a predictor of resistance as inhibition of phosphoAKT. Inhibition of phosphoERK displayed no relationship to gefitinib IC50. Phosphatase and tensin homolog (PTEN) was not present in the most resistant cell line but was present in representative sensitive cell lines suggesting that PTEN deletion is a mechanism underlying gefitinib resistance. Furthermore, a PI3K inhibitor in combination with gefitinib appeared more effective than the agents alone in the MTT assay. Conclusions: These results suggest that constitutively active AKT, possibly as a result of PTEN loss, is a mechanism of intrinsic gefitinib resistance in SCCHN. This resistance can be overcome through targeting of the PI3K/AKT pathway in combination with EGFR inhibition.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]