reports [29–33] 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 forma- tion 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 [34,35]. Thus, a more intensive study must be undertaken to determine the molecular relationship between vimentin and invadopodia struc- tures and cell motility and invasion. Microarray analysis revealed that Syk regulates a large number of genes in the TGFb1, NFkB, and EGFR pathways and that TGFb1 and VEGF are upregulated. These pathways constitute important regulators of cell invasion. In particular, TGFb-mediated EMT requires NF-kB [36]. 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 TGFb1 [37,38]. Both PI3K and NF-kB are reported to be inhibited by Syk in breast cancer cell lines [7] and they contribute to invadopodia formation [15] directly or indirectly. And, EGFR activity is downregulated by Syk as is the downstream kinase c-Src [4,8]. Thus, our results reveal that Syk is likely to control a TGFb1 signaling pathway in addition to EGFR/NFkB and their interacting genes (Figures S3–S4). 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 [39,40]. Materials and Methods Mice Ethics Statement. All animals were handled in strict accordance with good animal practice as defined by the Georgetown University Care and Use of Animals Committee (GUGUAC, protocol no. 07–020) and by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). Syk heterozygous (+/2) 129 background mice [41] were crossed with homozygous eGFP +/+ (Enhanced Green Fluorescent Protein) transgenic FVB mice and the progeny genotyped for Syk. eGFP mice were purchased from Jackson Laboratory (Bar Harbor, Maine). Syk +/2/eGFP +/+ were back crossed six times with homozygous eGFP +/+ mice. Homozygous eGFP +/+ mice were selected based on fluorescence intensity. These mice were genotyped for Syk and the Syk +/2/eGFP +/+ (referred to as Syk +/2) were used for this study. Genotypes of the mice were confirmed by PCR analysis using the oligomers 59-AGA GAA GCC CTG CCC ATG GAC-39, 59-CCT TGG GAA AAG CGC CTC CCC TAC CC -39 and 59-GTC CAG GTA GAC CTC Syk Loss in Breast Cells TTT GGG C-39. Methods for microscopic analysis of mammary gland morphologies are found in Supplementary File S1. Cell culture. MCF10A [42], MCF10AneoT [43], and MCF10DCIS.com [44] cell lines were acquired from Barbara Ann Karmanos Cancer Institute (obtained at passage number 174, 49, 30, respectively), passaged upon approaching confluence using standard techniques and were maintained in complete media (DMEM/F12 (50:50 mix) supplemented with 5% horse serum, 10 mM HEPES, 10 mg/ml insulin, 20 ng/ml epidermal growth factor, 0.5 mg/ml hydrocortisone, 100 ng/ml cholera toxin). Details and minor modifications of the methods for culture of cells on collagen I matrices, Matrigel, soft agar, and primary culture of mouse mammary epithelial cells and the assays used to quantify proliferation, migration, invasion, and morphologies are found in the Supplementary File S1 section. Microarray Analysis. Global gene expression profiles were measured using Affymetrix U133A 2.0 microarrays (with 22,283 probe sets) in MCF10A cell lines (control and Syk siRNA knockdown cells on collagen gels or plastic). Total RNA was extracted from cells seeded on collagen or plastic, using standard TRIZOL-based techniques. Fragmented cRNA was prepared as described in the Affymetrix Genechip Expression Analysis Technical Manual. Hybridization, washing, and imaging steps were performed in the Lombardi Comprehensive Cancer Center Macromolecular Shared Resource. Raw images were pre-processed using Microarray Suite 5.0 software (Affymetrix, Santa Clara, CA). For the preprocessing of the microarray data, we used SemiRMA [45] for background adjustment, loess for normalization and median polish for summarization. The preprocessed data (log- intensities) were then analyzed using a two-way ANOVA model with interaction to assess differential gene expression. The two factors are indicators for collagen coated dish (versus plastic dish) and Syk siRNA (versus empty vector). Due to the small number of replicates (3), we used empirical Bayes approach to borrow information across genes [46]. The method of Benjamini- Hochberg [47] was used to control FDR at 5%. Bioconductor packages ‘‘affy’’ and ‘‘limma’’ were used in the analysis. All data is MIAME compliant. Microarray data reported herein have been deposited at the NCBI Gene Expression Omnibus (http://www. ncbi.nlm.nih.gov/geo/) with the accession number GSE16200. Other statistical analysis. Results are shown as the mean 6 standard error of the mean (S.E.M.). Statistical significance was calculated by using the Student’s t-test and P,0.05 was accepted as a significant value. Supporting Information File S1 Additional material and methods. Found at: doi:10.1371/journal.pone.0007445.s001 (0.09 DOC) MB Table S1 Gene probes differentially regulated following Syk knockdown in MCF10A cells cultured on collagen. MCF10A cells cultured on collagen were subjected to microarray analysis to determine gene probe sets whose expression was differentially regulated following Syk knockdown. 1136 probe sets were significantly up- or down-regulated at the FDR level of 0.05 as described in Materials and Methods. Found at: doi:10.1371/journal.pone.0007445.s002 (0.18 MB PDF) Table S2 List of gene probe sets from merged networks 1, 2, and 10. Gene probe sets that were differentially regulated following Syk knockdown of MCF10A cells cultured on collagen (Supple- mentary Table 1) were submitted to Ingenuity for analysis. PLoS ONE | www.plosone.org 10 October 2009 | Volume 4 | Issue 10 | e7445