The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling
Dana M. Brantley-Sieders, … , Rebecca S. Muraoka-Cook, Jin Chen
Dana M. Brantley-Sieders, … , Rebecca S. Muraoka-Cook, Jin Chen
Published December 13, 2007
Citation Information: J Clin Invest. 2008;118(1):64-78. https://doi.org/10.1172/JCI33154.
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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 2

Loss of EphA2 expression impairs tumor formation and invasiveness in MMTV-Neu tumor cells.

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Loss of EphA2 expression impairs tumor formation and invasiveness in MMT...
(A) EphA2 expression was significantly diminished in MMTV-Neu tumor cells transduced with retroviruses expressing EphA2 siRNA sequences versus control siRNAs. Erk phosphorylation was reduced upon EphA2 knockdown. (B) Parental and control siRNA tumor cells formed large, irregularly shaped clusters with invasive protrusions (arrowheads) when cultured on Matrigel, whereas EphA2 siRNA–expressing cells formed smaller clusters with a rounded morphology and few protrusions, indicative of reduced invasiveness. Scale bar: 200 μm (top), 50 μm (bottom). We observed a significant decrease in colony size, as determined by calculating the average pixel area occupied by individual colonies, for cells expressing EphA2 siRNA relative to controls (P < 0.05; single-factor ANOVA). (C) Cultures stained with TO-PRO-3 iodide nuclear stain (blue) and anti–E-cadherin (green) were imaged by confocal microscopy. Control tumor cells formed multiacinar structures with invasive protrusions (arrowheads), whereas tumor cells expressing EphA2 siRNA sequences formed round, uniform acinar structures composed of a single layer of epithelial cells surrounding a central lumen (arrows). Scale bar: 20 μm. (D) Upon orthotopic transplantation into cleared fat pads of FVB recipient female mice, tumor cells expressing control siRNA sequences produced tumors of comparable volume to those generated by transplantation of parental cells at 5 weeks. Tumor cells expressing EphA2 siRNA sequences, however, either failed to form tumors or formed very small, nonpalpable tumors in a small fraction of animals (P < 0.05; single-factor ANOVA). Data are mean ± SEM.