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

BACKGROUND: TGFß 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-TGFß peptides in the control of angiogenesis elicited by conditional over-expression of TGFß. METHODS: We have inserted in human MCF7 mammary-cancer cells a mutated TGFß gene in a tetracycline-repressible vector to obtain conditional expression of mature TGFß upon transient transfection, evaluated the signaling pathways involved in TGFß-dependent endothelial cells activation and the efficacy of anti-TGFß peptides in the control of MCF7-TGFß-dependent angiogenesis. RESULTS: TGFß over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGFß-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 TGFß-stimulated EC resulted in impairment of GDF5 expression and of TGFß-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGFß antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGFß 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: TGFß 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 TGFß are controlled by anti-TGFß peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies.