Date of Award


Degree Type


Degree Name

Master of Science (MS)


Molecular Biology


Angiogenesis in NSCLC has been identified as important therapeutic target in combination with EGFR TKIs. However, only small incremental 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. I present experimental evidence that a subpopulation of NSCLC cells with EGFR TKI-induced EMT contributes toward the attenuation of the response to EGFR TKI therapy. One of the hallmarks of cancer is heterogeneity and I 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 and tumor-host interactions. While NSCLC cells with acquired EGFR TKI resistance and EMT phenotype did not exhibit growth advantage in vitro, a 50% epithelial EGFR TKI sensitive and 50% mesenchymal EGFR TKI resistant admix provided significant growth advantage in vivo assessed by caliper measurement. This preliminary result led us to hypothesize that changes in angiogenic growth factor expression during the EMT process might lead to the in vivo growth advantage I observed. To test the hypothesis, I utilized the Luminex multiplex assay system to quantify secreted growth factors, cytokines, and chemokines important in angiogenesis. I 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 EDN1. Using an in vitro tube formation assay, I showed that secreted VEGF-A and EDN1 in admix conditions work synergistically to promote endothelial cell differentiation. Furthermore, this synergistic effect can be attenuated by VEGFR2/EDNRA dual inhibition. Surprisingly, ectopic overexpression of EDN1 in EGFR- mutated HCC827 cells resulted in significant growth retardation in vivo. Informed by a literature search, I hypothesized that the presence of EDN1 in the tumor microenvironment contributes positively to EGFR TKI resistance, possibly through the vasoconstrictive property of EDN1. I observed that epithelial/mesenchymal admix tumors and ectopic overexpression of EDN1 in EGFR-mutated HCC827 cells conferred significantly more resistance to gefitinib in vivo. This result led us to hypothesize that EDN1 may reduce MVD in EGFR-mutated NSCLC tumors leading to poor EGFR TKI penetrance in vivo. I tested this through CD31 IHC staining and MVD calculation. I indirectly tested poor EGFR TKI penetrance by examining phosphorylated EGFR and found maintenance of the signal in admix and mesenchymal tumors. Taken together, I suggest that inhibition of the EDN1 signaling system may be an important component to a blood vascular-based approach to treatment of EGFR-mutation positive NSCLC.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.