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. 2008 Jan;118(1):64-78.
doi: 10.1172/JCI33154.

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

Dana M Brantley-Sieders  1 , 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
Affiliations

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

Dana M Brantley-Sieders et al. J Clin Invest. 2008 Jan.

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.

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Figures

Figure 1
Figure 1. EphA2 deficiency reduces mammary tumorigenesis, metastasis, proliferation, and vascularity in MMTV-Neu mice.
(A) Number of surface lung lesions was significantly reduced in EphA2–/– MMTV-Neu mice (P < 0.05; single-factor ANOVA). Data are mean ± SEM. (B) Top: Whole-mount mammary gland preparations (8 mo) revealed diminished hyperplasia in EphA2–/– glands relative to controls. Shown are an EphA2+/+ gland with pervasive epithelial hyperplasia (left) and an EphA2+/– gland with a small tumor (arrowhead; middle). Asterisks indicate inguinal lymph node. Bottom: H&E-stained mammary gland sections (8 mo) reveal reduced epithelial cell content in EphA2–/– MMTV-Neu tissue samples relative to controls. Scale bar: 250 μm. (C) Top: Mammary epithelial proliferation (PCNA+ nuclei; arrowheads), was significantly reduced (P < 0.05; 2-tailed, paired Student’s t test). Scale bar: 50 μm. Bottom: Mammary epithelial apoptosis (TUNEL+ nuclei; arrowheads) was not affected. (D) Top: Proliferation of primary mammary epithelial cells from EphA2–/– animals (BrdU incorporation; arrowheads) was reduced relative to EphA2+/+ cells (P < 0.05; 2-tailed, paired Student’s t test). Bottom: Apoptosis (TUNEL+ nuclei; arrowheads) was significantly increased in EphA2–/– primary mammary epithelial cells relative to controls (P < 0.05; 2-tailed, paired Student’s t test). Scale bar: 20 μm. (E) H&E-stained tumor sections (1 yr) demonstrate increased cystic degeneration and lumen formation in EphA2–/– tumors. Scale bar: 250 μm. (F) Decreased tumor cell proliferation (PCNA+ nuclei; arrowheads) was observed for EphA2–/– MMTV-Neu tumors compared with controls (P < 0.05; single-factor ANOVA). Scale bar: 50 μm. (G) Microvascular density (CD31+ vessels; arrowheads) was significantly reduced in EphA2–/– MMTV-Neu tumors relative to controls (P < 0.05; single-factor ANOVA). Scale bar: 100 μm.
Figure 2
Figure 2. Loss of EphA2 expression impairs tumor formation and invasiveness in MMTV-Neu tumor cells.
(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.
Figure 3
Figure 3. Elevated EphA2 expression in MCF10A.
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.
Figure 4
Figure 4. EphA2 is required for Ras/Erk activation and proliferation in the context of Neu/ErbB2-mediated neoplasia.
(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.
Figure 5
Figure 5. EphA2 is required for RhoA activation and tumor cell migration in the context of Neu/ErbB2-mediated malignancy.
(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.
Figure 6
Figure 6. EphA2 physically and functionally interacts with ErbB2.
(A) COS7 cells were transfected with plasmids for expression of EphA2 or/and ErbB2. EphA2 or ErbB2 was immunoprecipitated from cell lysates, and products were analyzed for ErbB2 or/and EphA2. Coexpression of EphA2 and ErbB2 was sufficient to permit coimmunoprecipitation. (B) Endogenous ErbB2 and EphA2 were coimmunoprecipitated with anti-EphA2 or anti-ErbB2 antibodies, respectively, in EphA2+/+ MMTV-Neu tumor cells that were untreated or treated with the chemical crosslinker DTSSP. The interaction detected was specific: EphA2 and ErbB2 were not immunoprecipitated by control IgG. Uniform input was validated by probing lysates for expression of EphA2 and ErbB2. (C) COS7 cells were transfected with plasmids for expression of EphA2 or ErbB2. EphA2 was immunoprecipitated from cell lysates, and products were analyzed for EphA2 expression and tyrosine phosphorylation. Coexpression of ErbB2 and EphA2 was sufficient to induce phosphorylation of EphA2 in COS7 cells in the absence of ephrin ligand stimulation. (D) Interaction between EphA2 and HER2 in MCF10A cells overexpressing HER2 was observed, as EphA2 and HER2 were coimmunoprecipitated with anti-EphA2 antibodies in HER2-overexpressing cells, but not in parental MCF10A cells. Elevated EphA2 phosphorylation was observed in MCF10A cells overexpressing HER2 relative to parental MCF10A cells, and treatment with the ErbB2 kinase inhibitor AG825 reduced EphA2 phosphorylation as well as ErbB2 phosphorylation in MCF10A cells overexpressing HER2.
Figure 7
Figure 7. EphA2 deficiency does not affect tumorigenesis, microvascular density, or growth regulatory signaling pathways in MMTV–PyV-mT tumors.
(A) Loss of EphA2 protein expression was confirmed by immunohistochemical staining. Scale bar: 50 μm. (B) We detected no change in MMTV–PyV-mT tumor microvascular density based on vWF staining (arrows indicate vWF+ blood vessels). Scale bar: 100 μm. (C) We did not observe any change in levels of GTP-bound active Ras or p-Erk in EphA2–/– MMTV–PyV-mT whole tumor extracts relative to controls, nor did we observe any change in levels of RhoA. Uniform loading was confirmed by immunoblotting for total Ras, total Erk, and tubulin. (D) We observed EphA2 overexpression and elevated phosphorylation in MMTV-Neu and MMTV–PyV-mT tumors relative to normal mammary tissue isolated from control FVB mice, with the highest levels observed in MMTV-Neu tumors. We also observed overexpression of ErbB2 and ephrin-A1 in both tumor types, with comparable ephrin-A1 expression in both tumor types and higher ErbB2 levels in MMTV-Neu tumors. Uniform loading was confirmed by immunoblot for actin. (E) We confirmed EphA2 overexpression specifically in epithelium by comparing EphA2 levels in PMEC lysates versus PMTCs derived from MMTV-Neu and MMTV–PyV-mT mice.
Figure 8
Figure 8. Treatment with an anti-EphA2 antibody inhibits tumor growth in MMTV-Neu but not MMTV–PyV-mT tumors.
(A) Treatment with anti–murine EphA2 antibody diminished EphA2 protein expression in tumor cells derived from MMTV-Neu and MMTV–PyV-mT mice. Tumor cells were treated with control IgG (10 μg/ml) or increasing concentrations of anti-EphA2 antibody for 48 hours. Uniform loading was confirmed by immunoblot for actin. Blots were stripped and reprobed with anti-EphA4 antibodies as a control for antibody specificity. (B) Cells derived from EphA2+/+ MMTV-Neu mice were orthotopically transplanted into the cleared fat pads of female FVB recipient mice. At 2 weeks following transplantation, mice were injected intraperitoneally with anti-EphA2 antibody or control IgG (10 mg/kg) twice weekly for 3 weeks. We observed a significant reduction in tumor volume in anti-EphA2–treated animals relative to control IgG–treated mice (P < 0.05; 2-tailed, paired Student’s t test). Data are mean ± SEM. (C) Tumor cell proliferation was significantly impaired in anti-EphA2–treated animals relative to controls (P < 0.05; single-factor ANOVA; arrowheads indicate PCNA+ nuclei). Scale bar: 50 μm. (D) EphA2 expression was significantly diminished in anti-EphA2–treated tumors relative to IgG controls, as assessed by immunohistochemistry and immunoblot. Blots were stripped and reprobed for actin expression to verify uniform loading. Scale bar: 50 μm. (E) We observed significantly reduced (P < 0.05; 2-tailed, paired Student’s t test) microvascular density in tumors isolated from anti-EphA2–treated mice relative to controls (arrowheads indicate vWF+ blood vessels). Scale bar: 100 μm. (F) Cells derived from MMTV–PyV-mT mice were orthotopically transplanted in the cleared fat pad of FVB female recipient mice and were treated with anti-EphA2 antibody or control IgG as described above. We observed no change in tumor volume between animals treated with anti-EphA2 antibody relative to control IgG-treated mice.
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