We purified Cerberus-Fc to homogeneity (Fig. As secreted antagonists, such as Cerberus, tightly regulate Nodal signaling during embryonic development, we undertook to produce human Cerberus, characterize its biochemical activities, and determine its effect on human breast malignancy Razaxaban cells. Using quantitative methods, we investigated the mechanism of Nodal signaling, we evaluated binding of human Cerberus to Nodal and other TGF? family ligands, and we characterized the mechanism of Nodal Spp1 inhibition by Cerberus. Using malignancy cell assays, we examined the ability of Cerberus to suppress aggressive breast malignancy cell phenotypes. We found that human Cerberus binds Nodal with high affinity and specificity, blocks binding of Nodal to its signaling partners, and inhibits Nodal signaling. Moreover, we showed that Cerberus profoundly suppresses migration, invasion, and colony forming ability of Nodal expressing and Nodal supplemented breast malignancy cells. Taken together, our studies provide mechanistic insights into Nodal signaling and Nodal inhibition with Cerberus and spotlight the potential value of Cerberus as anti-Nodal Razaxaban therapeutic. Introduction The Transforming Growth Factor-? (TGF?) family ligand Nodal is an essential regulator of vertebrate embryonic development that plays a critical role in formation of the primary body axes and in germ layer specification [1C3]. Beyond embryogenesis, the biological functions of Nodal appear to be limited and, in mammals, Nodal is usually thought to be largely absent from adult tissues, with exception of some adult stem cell populations and highly dynamic reproductive tissues [4C7]. However, a number of recent studies have shown that Nodal is usually re-expressed in various metastatic carcinomas, including melanoma and breast cancers, and that Nodal plays a critical role in promoting cancer progression [8C12]. For example, Nodal has been shown to be expressed by aggressive melanoma cells and contributes to their tumorigenicity and plasticity , Nodal levels correlate with invasive phenotypes in several breast malignancy cell lines [4, 10, 12], and Nodal is usually significantly overexpressed in tissue samples from patients diagnosed with advanced stage, invasive breast disease . Nodal knockdown, pharmacologic inhibition of Nodal signaling, and Nodal blockade with polyclonal antibodies or with Embryonic Stem Cell (ESC) conditioned medium have been shown to suppress the invasive and tumorigenic phenotype of Nodal expressing, melanoma and breast malignancy cells and [4, 8C10, 12C14]. Thus, Nodal is usually a potential therapeutic target in treatment of melanoma and breast cancers. However, Nodal inhibition is currently not a feasible clinical option, as existing small molecule inhibitors suffer from poor bioavailability and/or inadequate specificity [15, 16], and function-blocking anti-Nodal monoclonal antibodies have yet to be identified. During fish, frog, chick and mouse embryonic development, Nodal signaling is usually regulated by the secreted proteins Lefty and Cerberus . Both Lefty and Cerberus co-Immunoprecipitate Razaxaban (co-IP) with Nodal and antagonize Nodal signaling [17C23]. In addition, Lefty blocks Nodal receptor complex formation . Thus, it has been suggested that these embryonic Nodal-signaling antagonists could serve as Nodal inhibitors and potential anti-Nodal therapeutics . Indeed, Lefty purified from stem cell conditioned medium inhibited the colony forming ability of Nodal-expressing human melanoma cells and decreased tumor cell proliferation and increased tumor cell apoptosis when injected into tumors created from Nodal-expressing human melanoma cells . In contrast to Lefty, the embryonic Nodal antagonist Cerberus is usually less well comprehended and its molecular role during development as well as its potential as Nodal inhibitor in cancers have yet to be explored. We therefore undertook to elucidate, using purified, recombinant human proteins, the mechanism of Nodal signaling and Cerberus inhibition, and to characterize biological activities of human Cerberus in several human breast malignancy cell lines. Like all users of the TGF? family, Nodal signals Razaxaban by binding the extracellular domains of type I and type II receptor kinases, thus initiating a phosphorylation cascade that leads to Smad-2/3 mediated expression of Nodal target genes [25C31]. In addition, Nodal signaling during development requires membrane-anchored co-receptors [5, 26, 32, 33] (Fig. 1). Here, using human proteins, we recognized receptors and co-receptors that associate with.