The progression cell line series of MCF10A, MCF10AneoT, and DCIS.com exhibited a progressive loss of Syk. This result mirrors the loss of Syk observed in breast tissues from patients 
. Strikingly, knockdown of Syk using either siRNA or shRNA consistently enhanced proliferation, motility, invasion, and anchorage-independent growth in each of these cell lines along the progression series to DCIS.com, a cell line modeling ductal carcinoma in situ (DCIS). Thus, changes in levels of Syk potently mediate their effects on these activities and suggest that Syk must be present in epithelial cells at all stages of progression including benign tissues in order to suppress invasive, tumor-like cell behavior. The results in human breast epithelial cells are supported by the results in the mouse mammary gland where loss of one allele had an impressive effect on mammary ductal structure and ultimately on carcinoma development. In both human and mouse epithelial cells, we demonstrated increased cellular proliferation and invasion. The mouse heterozygote model further demonstrates that partial systemic loss of Syk can result in hyperplastic growth, tumor formation, and in some cases metastasis. The results on Syk knockdown in MCF10A cells support the interpretation that Syk is required in the epithelial cells of normal mouse mammary glands to control proper growth and invasion during puberty although it is possible that additional factors contribute since Syk is present in other tissues including immune cells and endothelium 
Multiple lines of evidence suggest that loss of Syk triggers EMT. EMT-related phenotypic changes are evidenced by change in cellular morphology and size in Matrigel and collagen I matrix. In addition, vimentin expression was strongly up-regulated following loss of Syk in the MCF10A cell lines. Vimentin loss is associated with the invasive and mesenchymal phenotype observed following EMT in multiple breast cancer cell types 
. Our observations of spontaneous EMT and vimentin upregulation in subconfluent cultures of MCF10a are consistent with previous reports 
and we now demonstrate that both can be suppressed by Syk. Interestingly, although vimentin expression in cell lines is correlated with their invasive capacity, vimentin filament expression was not correlated with invadopodia formation and degradation in individual cells. This is consistent with the report that although vimentin is associated with early sites of formation of podosomes in smooth muscle and osteoclasts, it does not seem to be directly involved in podosome formation and vimentin does not localize at the puncta of actin 
. Thus, a more intensive study must be undertaken to determine the molecular relationship between vimentin and invadopodia structures and cell motility and invasion.
Microarray analysis revealed that Syk regulates a large number of genes in the TGFβ1, NFκB, and EGFR pathways and that TGFβ1 and VEGF are upregulated. These pathways constitute important regulators of cell invasion. In particular, TGFβ-mediated EMT requires NF-κB 
. MCF10CA1, a tumor forming cell line derived from MCF10A, has been reported to form invadopodia and undergo EMT in a process requiring PI3K and c-Src in response to TGFβ1 
. Both PI3K and NF-κB are reported to be inhibited by Syk in breast cancer cell lines 
and they contribute to invadopodia formation 
directly or indirectly. And, EGFR activity is downregulated by Syk as is the downstream kinase c-Src 
. Thus, our results reveal that Syk is likely to control a TGFβ1 signaling pathway in addition to EGFR/NFκB and their interacting genes (Figures S3
). Thus, Syk potentially regulates at least three major signaling pathways to suppress proliferation and invasion. These pathways have important functions in the development and maintenance of the normal mammary gland and their disruption gives rise to EMT, invadopodia formation, and cancer progression. Consequently, the significance of the overlap between invadopodia-related proteins and cell surface stem cell markers (CD44 and CD49F) is not known but is highly suggestive of the involvement of invadopodia cell surface proteins in stem/progenitor cell function during development.
In conclusion, the present study demonstrates for the first time that the presence of wild type levels of Syk in normal breast epithelial cells is critical to suppress proliferation and invasion, and that partial loss promotes hyperplasia and the eventual formation of mammary tumors in vivo
. The significance of oral or locally applied Syk inhibitors for breast cancer risk is of critical concern in the context of the current development of agents targeting Syk for therapeutic treatment of allergy, asthma and autoimmune disease