Tumor Suppressor Function of Syk in Human MCF10A In Vitro and Normal Mouse Mammary Epithelium In Vivo You Me Sung 1 , Xuehua Xu1, Junfeng Sun 2 , Duane Mueller2, Kinza Sentissi1, Peter Johnson1, Elana Urbach1, Franc¸ oise Seillier-Moiseiwitsch2, Michael D. Johnson1, Susette C. Mueller1* 1 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C., United States of America, 2 Department of Biostatistics, Bioinformatics and Biomathematics, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D. C., United States of America Abstract The normal function of Syk in epithelium of the developing or adult breast is not known, however, Syk suppresses tumor growth, invasion, and metastasis in breast cancer cells. Here, we demonstrate that in the mouse mammary gland, loss of one Syk allele profoundly increases proliferation and ductal branching and invasion of epithelial cells through the mammary fat pad during puberty. Mammary carcinomas develop by one year. Syk also suppresses proliferation and invasion in vitro. siRNA or shRNA knockdown of Syk in MCF10A breast epithelial cells dramatically increased proliferation, anchorage independent growth, cellular motility, and invasion, with formation of functional, extracellular matrix-degrading invadopodia. Morphological and gene microarray analysis following Syk knockdown revealed a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodial cell surface markers CD44, CD49F, and MMP14. These results support the role of Syk in limiting proliferation and invasion of epithelial cells during normal morphogenesis, and emphasize the critical role of Syk as a tumor suppressor for breast cancer. The question of breast cancer risk following systemic anti-Syk therapy is raised since only partial loss of Syk was sufficient to induce mammary carcinomas. Citation: Sung YM, Xu X, Sun J, Mueller D, Sentissi K, et al. (2009) Tumor Suppressor Function of Syk in Human MCF10A In Vitro and Normal Mouse Mammary Epithelium In Vivo. PLoS ONE 4(10): e7445. doi:10.1371/journal.pone.0007445 Editor: Syed A. Aziz, Health Canada, Canada Received August 14, 2009; Accepted September 8, 2009; Published October 15, 2009 Copyright: 2009 Sung et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These studies were supported by the Susan G. Komen Foundation Postdoctoral fellowship BCTR70506 to Y.M.S, Department of Defense Idea award DAMD17-00-1-0273 and U.S. Public Health Service Grant NIH/NCI 9R01 CA112673 to S.C.M. We also thank the Histopathology and Tissue, Microscopy and Imaging, Tissue Culture, Biostatistics, Research Computing, and Flow Cytometry Shared Resources of Lombardi Comprehensive Cancer Center, which are partially supported by U.S. Public Health Service Grant 1P30-CA-51008 (Cancer Center Support Grant to Lombardi Cancer Center), 1S10 RR15768-01, and 1 S10 RR019291- 01A2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: muellers@georgetown.edu Introduction Understanding the normal function of Syk in the developing and adult breast, its upstream activators and downstream effectors, and epigenetic and genetic factors that cause its loss are vital for breast cancer prognosis, prediction and treatment. However, Syk plays an integral role in immune cell development and activation and it has been identified as a drug target for autoimmune disease, allergy and asthma. Syk is a tumor suppressor in breast cancer and may well also be in gastric cancer and melanoma, although it may be a tumor promoter in some invasive head and neck cancers and certain lymphomas [for review, [1] and [2,3]]. A complete understanding of the role of Syk as a tumor suppressor is vital for assessing breast cancer risk in patients who might be candidates for therapeutic suppression of Syk to control immune-related disease. Syk mRNA is increasingly lost from normal adjacent to hyperplastic to ductal carcinoma in situ (DCIS) to invasive tumors compared with normal, disease free breast epithelium [4]. The gene is hypermethylated in invasive breast cancer and loss of Syk has been tied to poor outcome in breast cancer [for review, [1]]. Functional studies in nude mice and in vitro illustrate the role of Syk in suppressing motility, invasion, and metastasis [1,5]. Syk has been shown to block breast cancer cell migration and chemoinva- sion via mechanisms involving down regulation of secreted growth factor GRO-1 [6] and suppression of NF-kB, PI3K [7], Src [4], and EGFR signaling pathways [8] [see also for review, [1]]. Studies on the role of Syk in cancer to date have all been conducted using tumor cell lines with reintroduction of Syk into highly invasive Syk negative cell lines or interference with endogenous Syk in otherwise non-invasive tumor cells. However, the normal function of Syk in mammary epithelium has not been addressed, nor have experiments been performed to determine the consequences of Syk loss from normal epithelial cells. MCF10A immortalized breast epithelial cells and cells derived from them serve as an in vitro model system to understand normal epithelial function and development [9,10]. MCF10A, MCF10AneoT, and DCIS.com form a series with which to model early cancer progression [11]. MCF10A are Syk positive, but the Syk status or functionality in this progression series is not known. The mouse mammary gland is another system to model normal epithelial behavior. The branching network of epithelial ducts arises from the combination of proliferation and epithelial ductal invasion through the fat pad via the formation of terminal end buds (TEB), TEB bifurcation and lateral branching, and ductal elongation. Unfortunately, nothing is known concerning Syk function in the adult or developing breast. Syk knockout mice have been PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7445