Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC
Katherine R. Amato, … , Nathanael S. Gray, Jin Chen
Katherine R. Amato, … , Nathanael S. Gray, Jin Chen
Published April 8, 2014
Citation Information: J Clin Invest. 2014;124(5):2037-2049. https://doi.org/10.1172/JCI72522.
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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 2

EphA2 deficiency results in increased apoptosis in KrasG12D tumors.

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EphA2 deficiency results in increased apoptosis in KrasG12D tumors. (...
(A) H&E-stained lung sections (25 weeks) showing tumors derived from KrasG12DEphA2–/– mice relative to those derived from KrasG12DEphA2+/+ mice. Scale bar: 200 μm. (B) Loss of EPHA2 protein expression in tumors was confirmed by immunohistochemistry (IHC). Scale bar: 50 μm. (C) Apoptosis in tumor sections was measured by the TUNEL assay. TUNEL+ nuclei (red) are indicated with arrowheads. Scale bar: 50 μm. (D) Apoptosis was quantified as a percentage of TUNEL-positive nuclei relative to the total nuclei. Apoptosis index is presented as average percentage of TUNEL-positive nuclei per total nuclei ± SEM (n = 6 per genotype). (E) Tumor cell proliferation was assessed by PCNA immunohistochemistry. Arrowheads indicate representative proliferating nuclei. Scale bar: 50 μm. (F) Proliferation was quantified by assessing the total number of PCNA+ nuclei (brown) compared with the total nuclei. Proliferation index is presented as average percentage of PCNA-positive nuclei per total nuclei ± SEM (n = 6 per genotype). (G) Tumor vasculature was assessed by vWF immunofluorescence (green). Arrowheads indicate tumor microvessels. Scale bar: 50 μm. (H) Microvessels in the tumor were quantified by measuring vWF+ pixels in each tumor field ± SEM (P = 0.07) (n = 6 per genotype). **P < 0.01.