Figure 6.

Molecular targeting of Aurora-A kinase activity in vivo restores an epithelial phenotype and suppresses breast cancer metastases. (a) Tumor imaging in live animals of vMCF-7^ΔRaf-1 xenografts expressing the firefly luciferase reporter lentivector. Following 8 weeks growth, tumor xenografts were treated with an empty shRNA vector (upper row) and an shRNA targeting Aurora-A (lower row) and imaged at 0, 2 and 4 weeks after treatment. Paraffin sections of xenograft tumors showing hematoxylin and eosin (H&E) staining of primary tumors and lung tissue. (b) Graph showing the area of tumor xenografts growth using NIH Image J program from three independent experiments. (c) Graph showing that nonmetastatic cells (vMCF-7^ΔRaf-1 1GX-NM) derived from tumor xenografts treated with shRNA Aurora-A decreased the percentage of CD24^–/low cells compared with control metastatic cells (vMCF-7^ΔRaf-1 1GX-M). The percentage of CD24^–/low cells was detected by FACS analysis from three independent experiments (±s.d). (d) Immunoblot analysis of vMCF-7^ΔRaf-1 1GX-M and vMCF-7^ΔRaf-1 1GX-NM cells showing suppression of EMT and restoration of an epithelial phenotype in vMCF-7^ΔRaf-1 1GX-NM cells. (e) Immunofluorescence analysis showing expression of epithelial markers E-cadherin and β-catenin, lack of vimentin and inhibition of nuclear SMAD5 phosphorylation in vMCF-7^ΔRaf-1 1GX-NM cells. E-cadherin and p-SMAD5 were labeled in red, β-catenin and vimentin were labeled in green and DNA was labeled in blue with Hoechst dye.