Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC
Katherine R. Amato, Shan Wang, Andrew K. Hastings, Victoria M. Youngblood, Pranav R. Santapuram, Haiying Chen, Justin M. Cates, Daniel C. Colvin, Fei Ye, Dana M. Brantley-Sieders, Rebecca S. Cook, Li Tan, Nathanael S. Gray, Jin Chen
Katherine R. Amato, Shan Wang, Andrew K. Hastings, Victoria M. Youngblood, Pranav R. Santapuram, Haiying Chen, Justin M. Cates, Daniel C. Colvin, Fei Ye, Dana M. Brantley-Sieders, Rebecca S. Cook, Li Tan, Nathanael S. Gray, Jin Chen
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Research Article Oncology

Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC

Abstract

Genome-wide analyses determined previously that the receptor tyrosine kinase (RTK) EPHA2 is commonly overexpressed in non–small cell lung cancers (NSCLCs). EPHA2 overexpression is associated with poor clinical outcomes; therefore, EPHA2 may represent a promising therapeutic target for patients with NSCLC. In support of this hypothesis, here we have shown that targeted disruption of EphA2 in a murine model of aggressive Kras-mutant NSCLC impairs tumor growth. Knockdown of EPHA2 in human NSCLC cell lines reduced cell growth and viability, confirming the epithelial cell autonomous requirements for EPHA2 in NSCLCs. Targeting EPHA2 in NSCLCs decreased S6K1-mediated phosphorylation of cell death agonist BAD and induced apoptosis. Induction of EPHA2 knockdown within established NSCLC tumors in a subcutaneous murine model reduced tumor volume and induced tumor cell death. Furthermore, an ATP-competitive EPHA2 RTK inhibitor, ALW-II-41-27, reduced the number of viable NSCLC cells in a time-dependent and dose-dependent manner in vitro and induced tumor regression in human NSCLC xenografts in vivo. Collectively, these data demonstrate a role for EPHA2 in the maintenance and progression of NSCLCs and provide evidence that ALW-II-41-27 effectively inhibits EPHA2-mediated tumor growth in preclinical models of NSCLC.

Authors

Katherine R. Amato, Shan Wang, Andrew K. Hastings, Victoria M. Youngblood, Pranav R. Santapuram, Haiying Chen, Justin M. Cates, Daniel C. Colvin, Fei Ye, Dana M. Brantley-Sieders, Rebecca S. Cook, Li Tan, Nathanael S. Gray, Jin Chen

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Figure 4

EPHA2 knockdown leads to an increase in apoptosis in NSCLC cell lines.

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EPHA2 knockdown leads to an increase in apoptosis in NSCLC cell lines. ...
(A) Cells were treated with scrambled or EPHA2-specific siRNA for 72 hours. Apoptosis was detected via the ApopTag TUNEL assay. Graph represents 3 independent experiments, and data are presented as the percentage of TUNEL-positive nuclei of total nuclei ± SEM. (B) Western blotting of H2009 and H358 cells treated with scrambled or siEPHA2 for 72 hours with 5 μg/ml TRAIL added during the final 24 hours after transfection. Cl., cleaved (caspase-3 or PARP). (C) Apoptosis was measured by quantifying histone-associated DNA fragments using a Cell Death ELISA Kit. Cells were treated with scrambled or siEPHA2 for 72 hours before the assay. All 6 cell lines exhibited a statistically significant increase in apoptosis in the cells treated with siEPHA2 compared with the scrambled controls. Experiments were repeated 3 times, and data are presented as average absorbance unit (AU) ± SEM. (D) H2009 and H358 cells were treated with scrambled or siEPHA2 for 72 hours. Cells were starved for 24 hours and stimulated with 10% serum for 10 minutes before lysis. Shown are representative immunoblots in which phosphorylation levels of signaling molecules were detected using anti-phospho antibodies and EPHA2 expression was detected by an anti-EPHA2 antibody. *P < 0.05, **P < 0.01.