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 5

EphA2 is required for RhoA activation and tumor cell migration in the context of Neu/ErbB2-mediated malignancy.

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EphA2 is required for RhoA activation and tumor cell migration in the co...
(A) EphA2–/– PMTCs displayed significantly reduced migration in response to growth media supplemented with 10% serum compared with EphA2+/+ PMTCs in transwell migration assays (P < 0.05; 2-tailed, paired Student’s t test). (B) RhoA activity, as measured by effector pulldown assay of GTP-bound RhoA in tumor cell lysates and in whole tumor extracts by GST-Rhotekin Rho-binding domain, was reduced in EphA2–/– PMTCs and intact tumors relative to EphA2+/+ cells and tumors. We also observed a decrease in total RhoA protein levels in EphA2–/– MMTV-Neu tumor cells and in whole tumor extracts relative to EphA2+/+ controls. We observed no change in GTP-bound, activated Rac, or total Rac protein levels in tumor cell lysates from EphA2–/– or EphA2+/+ PMTCs. (C) For rescue experiments, EphA2–/– MMTV-Neu primary tumor cells were transduced with adenoviruses expressing constitutively active RhoA (Q63L) or control β-gal 48 hours prior to migration assay. Expression of constitutively active RhoA restored serum-induced migration of EphA2–/– tumor cells to levels comparable to those observed in tumor cells derived from EphA2+/+ animals, while control β-gal had no effect (P < 0.05, EphA2–/– Ad–β-gal versus EphA2+/+ and EphA2–/– Ad-Rho; single-factor ANOVA). Expression of adenoviral transgenes was confirmed by immunoblot assays.