Abstract B14: Erlotinib-cisplatin combination inhibits growth and angiogenesis via c-MYC and HIF-1a in EGFR mutated lung cancer
Authors: Lee Jasmine G. , Wu Reen 
Published: December 2015

Abstract
Introduction: Combination treatment for non-small cell lung cancer (NSCLC) is becoming more popular due to the anticipation that it may be more effective than single drug treatment. In addition, there are efforts to genetically screen patients for specific mutations in light of attempting to administer specific anticancer agents that are most effective. In this study, we evaluate the anticancer and anti-angiogenic effects of low dose erlotinib-cisplatin combination in NSCLC in vitro and in vivo.
Method: Human lung adenocarcinoma cell lines (A549, NCI-H292, H1650, PC9, H3255 and HCC827) were utilized. Each cell line had four treatment groups: control, erlotinib, cisplatin, and erlotinib -cisplatin combination. Alamar Blue Assay was utilized to evaluate for in vitro cell death following two day treatments. PC9 cells were used for in vivo experiments. Erlotinib and/or cisplatin treatments were injected directly into the tumor and tumor measurements were made every 2 days for a total of 14 days. Western blot analysis was performed to measure the level of VEGF, CD31, c-MYC, and HIF-1a for both in vitro and in vivo samples. Hematoxylin and eosin (H&E) staining and 3,3'-Diaminobenzidine (DAB) staining for CD31 were done on tumor samples. In vivo angiogenesis assay was tested though the use of the matrigel plug assay. Microvessel density quantification was quantified using ImageJ, and the percentage area of CD31 was calculated by imaging four different 20x high power fields through hotspot method.
Results: In NSCLC cells harboring epidermal growth factor receptor (EGFR) mutations, combination erlotinib-cisplatin treatment led to synergistic cell death, but there minimal efficacy in NSCLC cells with wild-type EGFR. In xenograft models, combination treatment also demonstrated greater inhibition of tumor growth compared to individual treatment. The anti-tumor effect observed was secondary to the targeting of angiogenesis, evidenced by decreased VEGF levels, and decreased levels of CD31 and microvessel density. Combination treatment targets angiogenesis through down-regulation of the c-MYC/HIF-1a pathway. In fact, cell lines with EGFR exon 19 deletions expressed high basal levels of c-MYC and HIF-1a and correlate with robust responses to combination treatment.
Conclusion: These results suggest that low dose erlotinib-cisplatin combination exhibits its anti-tumor activity by targeting angiogenesis through the modulation of the c-MYC/HIF-1a/VEGF pathway in NSCLC with EGFR exon 19 deletions. These findings may have significant clinical implications in patients with tumors harboring EGFR exon 19 deletions as they may be particularly sensitive to this regimen.
Citation Format: Jasmine G. Lee, Reen Wu. Erlotinib-cisplatin combination inhibits growth and angiogenesis via c-MYC and HIF-1a in EGFR mutated lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B14.