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.