Abstract 20: Inhibition of mutant EGFR in NSCLC promotes endothelin-1-mediated NSCLC disease progression and angiogenesis

Despite recent advances in the treatment of NSCLC targeting of EGFR kinase domain mutations with tyrosine kinase inhibitors (TKIs), work needs to be done to reduce morbidity and improve survival for NSCLC patients. In NSCLC, tumor angiogenesis has been identified as important therapeutic target in combination with EGFR TKIs. However, only small advancements have been made for the use of angiogenesis inhibitors in NSCLC and it remains elusive why the inhibition of VEGF-mediated neovascularization is not therapeutically efficacious. We present evidence that a subpopulation of NSCLC cells with the EGFR TKI-induced epithelial to mesenchymal transition (EMT) contributes to the attenuation of response to anti-VEGF/VEGFR therapy. One of the hallmarks of cancer is heterogeneity and we have previously demonstrated that tumor heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations gives rise to divergent resistance mechanisms in response to treatment. In vivo admix models are instructive in studying intratumoral heterogeneity and in elucidating therapeutic responses. While NSCLC cell with acquired EGFR TKI resistance and EMT phenotype did not exhibit growth advantage in vivo, a 50% epithelial EGFR TKI sensitive and 50% mesenchymal EGFR TKI resistant admix provided significant growth advantage in vivo assessed by caliper measurement. Moreover, the admix tumors are resistant to EGFR TKI treatment. Interestingly, short-term in vitro co-culture of epithelial and mesenchymal cells did not provide a proliferative/growth advantage. The preliminary result led us to hypothesize that the epithelial-mesenchymal admix helps to create a tumor-host niche that is suitable for EGFR TKI resistance. To this end, we utilized the Luminex multiplex assay system to quantify secreted growth factors, cytokines, and chemokines. We have discovered that epithelial EGFR TKI sensitive cells secrete a significant amount of VEGF-A and cells with acquired/transient EGFR TKI resistance with an EMT phenotype secrete substantial amount of endothelin-1 (EDN1). Using an in vitro tube formation assay, we showed that secreted VEGF-A and EDN1 in admix conditions work synergistically to promote angiogenesis. Ectopic overexpression of EDN1 in EGFR-mutated HCC827 cells and predominantly secreting VEGF-A conferred significant resistance to gefitinib in vivo compared to control HCC827 cells. Importantly, the increased HUVEC cell tube formation in conditioned media from admix cells rich in