The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling
J. Clin. Invest. Dana M. Brantley-Sieders, et al. 118:64 doi:10.1172/JCI33154 [Go to this article.]

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.