GDF5 Regulates TGFβ-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and In Vivo Control by Anti-TGFβ Peptides

Background: TGF beta overproduction in cancer cells is one of the main characteristics of late tumor progression being implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGF beta peptides in the control of angiogenesis elicited by conditional over-expression of TGF beta. Methods: We have inserted in human MCF7 mammary-cancer cells a mutated TGF beta gene in a tetracycline-repressible vector to obtain conditional expression of mature TGF beta upon transient transfection, evaluated the signaling pathways involved in TGF beta-dependent endothelial cells activation and the efficacy of anti-TGF beta peptides in the control of MCF7-TGF beta-dependent angiogenesis. Results: TGF beta over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGF beta-transfected MCF7 stimulated angiogenesis in vivo and in vitro by subsequent activation of SMAD2/3 and SMAD1/5 signaling in endothelial cells, as well as SMAD4 nuclear translocation, resulting in over-expression of the pro-angiogenic growth and differentiation factor-5 (GDF5). Inhibition or silencing of GDF5 in TGF beta-stimulated EC resulted in impairment of GDF5 expression and of TGF beta-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGF beta antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGF beta in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, GDF5 and uPAR overexpression. Antagonist peptides and anti-GDF5 antibodies efficiently inhibited in vitro and in vivo angiogenesis. Conclusions: TGF beta produced by breast cancer cells induces in endothelial cells expression of GDF5, which in turn stimulates angiogenesis both in vitro and in vivo. Angiogenesis activation is rapid and the involved mechanism is totally opposed to the old and controversial dogma about the AKL5/ALK1 balance. The GDF-dependent pro-angiogenic effects of TGF beta are controlled by anti-TGF beta peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies.