The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling
Dana M. Brantley-Sieders, Guanglei Zhuang, Donna Hicks, Wei Bin Fang, Yoonha Hwang, Justin M.M. Cates, Karen Coffman, Dowdy Jackson, Elizabeth Bruckheimer, Rebecca S. Muraoka-Cook, Jin Chen
Dana M. Brantley-Sieders, Guanglei Zhuang, Donna Hicks, Wei Bin Fang, Yoonha Hwang, Justin M.M. Cates, Karen Coffman, Dowdy Jackson, Elizabeth Bruckheimer, Rebecca S. Muraoka-Cook, Jin Chen
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Research Article Oncology

The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling

Abstract

Overexpression of the receptor tyrosine kinase EPH receptor A2 (EphA2) is commonly observed in aggressive breast cancer and correlates with a poor prognosis. However, while EphA2 has been reported to enhance tumorigenesis, proliferation, and MAPK activation in several model systems, other studies suggest that EphA2 activation diminishes these processes and inhibits the activity of MAPK upon ligand stimulation. In this study, we eliminated EphA2 expression in 2 transgenic mouse models of mammary carcinoma. EphA2 deficiency impaired tumor initiation and metastatic progression in mice overexpressing ErbB2 (also known as Neu) in the mammary epithelium (MMTV-Neu mice), but not in mice overexpressing the polyomavirus middle T antigen in mammary epithelium (MMTV–PyV-mT mice). Histologic and ex vivo analyses of MMTV-Neu mouse mammary epithelium indicated that EphA2 enhanced tumor proliferation and motility. Biochemical analyses revealed that EphA2 formed a complex with ErbB2 in human and murine breast carcinoma cells, resulting in enhanced activation of Ras-MAPK signaling and RhoA GTPase. Additionally, MMTV-Neu, but not MMTV–PyV-mT, tumors were sensitive to therapeutic inhibition of EphA2. These data suggest that EphA2 cooperates with ErbB2 to promote tumor progression in mice and may provide a novel therapeutic target for ErbB2-dependent tumors in humans. Moreover, EphA2 function in tumor progression appeared to depend on oncogene context, an important consideration for the application of therapies targeting EphA2.

Authors

Dana M. Brantley-Sieders, Guanglei Zhuang, Donna Hicks, Wei Bin Fang, Yoonha Hwang, Justin M.M. Cates, Karen Coffman, Dowdy Jackson, Elizabeth Bruckheimer, Rebecca S. Muraoka-Cook, Jin Chen

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Figure 4

EphA2 is required for Ras/Erk activation and proliferation in the context of Neu/ErbB2-mediated neoplasia.

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EphA2 is required for Ras/Erk activation and proliferation in the contex...
(A) Proliferation of PMTCs isolated from EphA2–/– animals, as assessed by nuclear incorporation of BrdU (arrowheads), was reduced relative to EphA2+/+ cells. For rescue experiments, PMTCs were transduced with adenoviruses expressing EphA2 or β-gal 48 hours prior to BrdU incorporation assay. Overexpression of EphA2 significantly elevated serum-induced proliferation relative to control (P < 0.05; 2-tailed, paired Student’s t test). Scale bar: 20 μm. Expression of adenoviral transgenes was confirmed by immunoblot. (B) Ras activity in unstimulated cells, as measured by effector pulldown assay of GTP-bound Ras by GST-Raf Ras-binding domain, was reduced in EphA2–/– PMTCs relative to control, as was Erk phosphorylation. Uniform loading was confirmed by immunoblotting for total Ras/Erk and actin. EphA2 deficiency and uniform expression of Neu/ErbB2 was confirmed by effector pulldown assay and immunoblotting for EphA2 and ErbB2. EphA2 was phosphorylated in unstimulated EphA2+/+ tumor cells, and no changes in ErbB2 phosphorylation were detected in EphA2+/+ versus EphA2–/– PMTCs. (C) Diminished Ras and Erk activity were confirmed in whole tumor extracts isolated from 3 independent EphA2+/+ or EphA2–/– tumors. (D) For rescue experiments EphA2–/– PMTCs were transduced with adenoviruses expressing Erk-1 or control βgal. Overexpression of Erk-1 in EphA2–/– PMTCs significantly elevated serum-induced proliferation relative to control (P < 0.05, EphA2–/– Ad–β-gal versus EphA2+/+ or EphA2–/– Ad-Erk-1; single-factor ANOVA). Expression of adenoviral transgenes was confirmed by immunoblot. (E) Treatment of EphA2+/+ PMTCs with the MEK inhibitor U0126 for 12 hours significantly inhibited serum-induced proliferation relative to vehicle control (P < 0.05, 5- and 10-μM U0126 versus vehicle). Inhibition of Erk phosphorylation by U0126 was confirmed by immunoblot.