Whole-cell protein lysates (20 g) from each clonal cell population (treated and untreated) were aliquoted into 96-well plates, luciferase assay reagent added to each well, and luciferase activity measured with a BMG FLUOstar Omega plate reader (Imgen Technologies). cell lines. Live-cell migration assays reveal that BMP4 induces breast cancer migration, which is usually effectively blocked by Chrdl1. We demonstrate that BMP4 also stimulated breast cancer cell invasion and matrix degradation, in part, through enhanced metalloproteinase 2 (MMP2) and MMP9 activity that is antagonized by Chrdl1. Finally, high Chrdl1 expression was associated with better clinical outcomes in patients with breast cancer. Together, our data reveal that Chrdl1 acts as a negative regulator of malignant breast cancer phenotypes through inhibition of BMP signaling. INTRODUCTION Breast cancer is usually a heterogeneous disease that can be subdivided into distinct molecular subtypes through the integration of gene expression and genomics data (1, 2). While ErbB2+ breast cancers are considered a poor-prognosis subtype (3), other signaling pathways can further modulate their malignant phenotypes. The transforming growth factor (TGF-) family is usually a prominent example that has been shown to enhance the migratory, invasive, and metastatic abilities of ErbB2+ breast cancer cells (4,C7). We have previously exhibited that this ShcA adaptor protein plays an important role, downstream of TGF- and ErbB2 signaling pathways, in mediating these cellular responses (8, 9). Loss of ShcA expression Clasto-Lactacystin b-lactone in ErbB2-expressing cells significantly reduced tumor growth, which was Clasto-Lactacystin b-lactone the result of reduced proliferation, diminished endothelial cell recruitment, and elevated apoptosis (9). In the present study, through the use of microarray based transcriptional profiling, we identified elevated levels of chordin-like 1 (Chrdl1) in ErbB2+ breast cancer cells following TGF- stimulation; however, this upregulation of Chrdl1 occurs only in the context of diminished ShcA levels. Bone morphogenetic proteins (BMPs) are secreted cytokines that belong to the TGF- family of proteins, and their aberrant expression is observed in numerous cancers, including breast cancer (10). However, much like the TGF- isoforms, there are conflicting reports on whether BMPs exert pro- or antitumorigenic effects on cancer cells (11, 12). In breast cancer, BMP4 has been shown to promote cancer cell migration and invasion (13,C16). Similarly, BMP7 induces breast cancer cell proliferation, migration/invasion, and metastasis (17, 18). Interestingly, BMP4 and BMP7 are the most frequently and most highly expressed family members in breast cancer (10, 14). Several factors influence BMP expression and activity. The BMP pathway can be negatively regulated by BMP antagonists, which are secreted proteins that bind BMP ligands and block their interactions with cognate cell surface receptors (19). A tight balance between BMP and BMP antagonist activity is required during development and normal tissue homeostasis in the adult. Disruption of this balance contributes to the progression of numerous diseases, including cancer (20). Chrdl1 is usually a secreted antagonist of BMP-mediated signaling via the Smad pathway, and it has previously been reported to predominantly inhibit BMP4-mediated signaling (21). While Chrdl1 function has been studied mainly in the context of development, little is known about its putative role in breast cancer. In the current study, we exhibited that, in the context of reduced ShcA signaling, Chrdl1 expression is upregulated in numerous breast cancer cells following TGF- stimulation. Through a series of experiments, we exhibited that Chrdl1 acts as an inhibitor of BMP4-induced migration and invasion. Moreover, Chrdl1 expression serves as a prognostic factor for better outcomes in patients with breast cancer. MATERIALS AND METHODS Generation of an inducible ShcA knockdown system in NMuMG-ErbB2 breast cancer cells. Retrovirus harboring the reverse Tet transactivator (rtTA) was generated using 293 vesicular stomatitis virus (VSV) packaging cells as previously described (22). NMuMG cells were transduced with virus and selected using 400 g/ml of neomycin-G418 (Wisent) to generate a pooled cell population expressing the rtTA. Clonal populations were subsequently established and characterized for doxycycline inducibility using a tetracycline responsive element (TRE)-luciferase reporter construct Clasto-Lactacystin b-lactone (Clontech). Briefly, transient retroviral infections were performed Clasto-Lactacystin b-lactone with the TRE-luciferase reporter; individual clones were subsequently split into two pools and left untreated or treated with 2 g/ml of doxycycline for 48 h. Cell lysates were prepared with luciferase assay cell lysis buffer from the TIE1 Promega luciferase assay system (catalog number E1500). Whole-cell protein lysates (20 g) from each clonal cell population (treated and untreated) were aliquoted into 96-well plates, luciferase assay reagent added to each well, and luciferase activity measured with a BMG FLUOstar Omega plate reader (Imgen Technologies). Luminescence readings were normalized to protein concentrations determined from the cell lysates, and induced luciferase expression was determined by plotting normalized luminescence values Clasto-Lactacystin b-lactone with and without doxycycline treatment. Populations that exhibited a 13-fold or greater induction in luciferase activity following doxycycline.