Bone Morphogenetic Protein-14 Regulates TGF-β-Dependent Angiogenesis in Breast Carcinoma MCF-7 Cells: In Vitro and in Vivo Control by Anti-TGF-β Peptides

Background: TGF-β overproduction by breast cancer cells is one of the main characteristics of the late phase of tumor progression and is implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGF-β peptides in the control of TGF-β angiogenesis. Methods: We have inserted in human MCF-7 mammary cancer cells a mutated TGF-β gene in a tetracycline-repressible vector to obtain conditional expression of mature TGF-β in the absence of tetracycline upon transient transfection and have evaluated the efficacy of anti-TGF-β peptides in the control of MCF-7 TGF-β -dependent angiogenesis. Results: TGF-β overexpression induced in MCF-7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGF-β-transfected MCF-7 stimulated angiogenesis in vivo and in vitro by subsequent activation of ALK5-SMAD2/3 and ALK1-SMAD1/5 signaling in endothelial cells (EC), as well as SMAD4 nuclear translocation, resulting in overexpression of the proangiogenic bone-morphogenetic-protein-14 (BMP14/GDF5). Antibody inhibition and siRNA silencing of BMP14 in TGF-β-stimulated EC resulted in impairment of BMP14 expression and of TGF-β-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGF-β antagonist peptides efficiently inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGF-β in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, BMP14 and uPAR overexpression. Antagonist peptides efficiently inhibited in vivo angiogenesis either when co-injected with tumor cells or upon systemic administration. Conclusions: These preclinical data provide a basis to support using anti–TGF-β peptides as a therapeutic agent for TGFβ–dependent breast cancer angiogenesis.