Abstract 4271: Epidermal growth factor receptor variant III (EGFRvIII) expression initiates slower proliferation and signals through AKT and STAT3 in immortalized keratinocytes.

Introduction: Expression of EGFRvIII has been reported in ∼40% of head and neck squamous cell carcinomas (HNSCCs). EGFRvIII lacks the ligand binding domain (exons 2-7), thus this receptor has demonstrated resistance to cetuximab (CTX), a monoclonal antibody against EGFR. Although ligand-independent activation likely drives downstream activation, EGFRvIII-specific pathways are currently unknown. To delineate the downstream effects of EGFRvIII and its contribution to EGFR inhibitor resistance, we transduced an immortalized human keratinocyte cell line (HaCaT), and Chinese hamster ovarian (CHO) cells, which lack EGFR expression to examine the isolated and contextual biological properties of EGFRvIII in vitro and in vivo. Methods: HaCaT and CHO cells were transduced with EGFRvIII, EGFR wild type (EGFR-WT), and an empty vector control. Proliferation of the transduced cells was determined using MTT. Subsequent sensitivity to CTX or gefitinib was determined by colony formation assay. Receptor activation and downstream signaling following ligand stimulation was assessed by Western blot. Subcutaneous hindflank xenografts of transduced HaCaTs were established in immunodeficient mice and resulting tumor growth was monitored. EGFRvIII expression from 30 HNSCC patient samples was also determined by RT-PCR. Results: HaCaT-EGFRvIII demonstrated slower proliferation than HaCaT-EGFR-WT. Ligand-stimulated EGFRvIII was also slower to activate, with EGFR reaching maximal phosphorylation at 30 min compared to 8 hr with EGFRvIII. We also observed that EGFRvIII preferentially signaled through AKT and STAT3, while EGFR utilized both AKT and MAPK. HaCaT-EGFR-WT xenografts exhibited rapid growth, with exponential growth initiating at 4 weeks while HaCaT-EGFRvIII demonstrated slow, linear phase growth at 12 weeks. In isolation, EGFRvIII exhibited a differential phosphorylation pattern compared to EGFR. Tyr 1068 and 1173 were both phosphorylated in EGFR and EGFRvIII, but Tyr 992 and 1086 were not activated in EGFRvIII. EGFR activation in CHO cells was reversible when treated with CTX and gefitinib, while EGFRvIII was only inhibited by gefitinib. However, HaCaT-EGFRvIII cells were not resistant to CTX in vitro. Utilizing RT-PCR, we determined EGFRvIII expression was lower (13%) than previously reported in HNSCC tumor samples. Conclusion: We determined EGFRvIII expression results in a slower proliferation rate in vitro and in vivo compared to EGFR overexpression. Additionally, EGFRvIII preferentially signals downstream through AKT and STAT3. In isolation, EGFRvIII is resistant to CTX inhibition, but does not confer CTX resistance in a fully competent keratinocyte model system. Ultimately, the clinical significance of EGFRvIII in HNSCC is unclear, as EGFR is always expressed in concert with EGFRvIII and its apparent incidence is low. Citation Format: Hiroyuki Ozawa, Ana Markovic, Jason Howard, Harry Quon, Jimena Perez, Robert Hughes, Christine H. Chung. Epidermal growth factor receptor variant III (EGFRvIII) expression initiates slower proliferation and signals through AKT and STAT3 in immortalized keratinocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4271. doi:10.1158/1538-7445.AM2013-4271