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 3

Elevated EphA2 expression in MCF10A.

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Elevated EphA2 expression in MCF10A. ER2 cells enhances cell proliferati...
ER2 cells enhances cell proliferation and invasiveness in vitro. (A) Parental MCF10A human breast cells and MCF10A.HER2 cells were transduced with adenoviruses (Ad) expressing EphA2 or control β-gal and plated on growth factor–reduced Matrigel to generate 3-dimensional spheroid cultures. After 10 days in culture, parental MCF10A cells and cells expressing Ad–β-gal formed small, round acinar structures, while MCF10A.HER2 cells formed larger colonies with irregular, invasive protrusions (arrows). Expression of Ad-EphA2 in MCF10A cells resulted in larger, irregular colonies, an effect that was amplified in MCF10A.HER2 cells (P < 0.05; single-factor ANOVA). Scale bar: 25 μm. (B) Cultures were stained with TO-PRO-3 iodide nuclear stain (red) and anti-Ki67 (green) and imaged by confocal microscopy. Confocal analysis revealed that parental and Ad–β-gal–transduced MCF10A formed uniform acinar structures composed of a single layer of epithelial cells surrounding a central lumen, while MCF10A.HER2 cells formed multiacinar structures with invasive protrusions (arrows) and a poorly defined lumen containing several cells. MCF10A cells transduced with Ad-EphA2 also formed multiacinar structures with a poorly defined lumen. Invasion and lumen filling were enhanced in MCF10A.HER2 cells overexpressing EphA2. Scale bar: 20 μm. EphA2 overexpression significantly enhanced proliferation (Ki67+ nuclei, arrows) within acinar structures formed by MCF10A and MCF10A.HER2 cells (P < 0.05; single-factor ANOVA). (C) Expression of adenoviral gene products and overexpression of ErbB2/HER2 in MCF10A.HER2 cells was confirmed by immunoblot, and uniform loading was verified by immunoblot for actin. Expression of p-Erk, total Erk, p-EphA2, and total EphA2 was also assessed by immunoblot.