Myoferlin Depletion in Breast Cancer Cells Promotes Mesenchymal to Epithelial Shape Change and Stalls Invasion Ruth Li1, William E. Ackerman, IV1, Cosmin Mihai4, Leonithas I. Volakis2, Samir Ghadiali2,3, Douglas A. Kniss1,2* 1 Laboratory of Perinatal Research, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, The Ohio State University, Columbus, Ohio, United States of America, 2 Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio, United States of America, 3 Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, United States of America, 4 Pacific Northwest National Laboratory, Richland, Washington, United States of America Abstract Myoferlin (MYOF) is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET). These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin) and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNAMYOF transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF. Citation: Li R, Ackerman WE IV, Mihai C, Volakis LI, Ghadiali S, et al. (2012) Myoferlin Depletion in Breast Cancer Cells Promotes Mesenchymal to Epithelial Shape Change and Stalls Invasion. PLoS ONE 7(6): e39766. doi:10.1371/journal.pone.0039766 Editor: Wanjin Hong, Institute of Molecular and Cell Biology, Singapore Received April 23, 2012; Accepted May 30, 2012; Published June 27, 2012 Copyright: 2012 Li 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: This work was supported by the Ohio State University Perinatal Research and Development Fund (DAK), NIH K08 HD49628 (WEA), and the National Science Foundation Nanoscience Science and Engineering Center grant #EEC-0425626 (RL) and Chemical, Bioengineering, Environmental, and Transport Systems (CBET) #1134201 (LV, DAK, SG). 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: kniss.1@osu.edu Introduction Breast cancer is the second leading cause of cancer mortality in women [1], with the majority of the deaths due to metastatic rather than localized disease [2]. Unrestrained cell division elicited by somatic and/or germline mutations in several oncogenes and tumor suppressor genes such as TP53 and Rb, and resistance to programmed cell death are hallmarks of tumorigenesis [3,4]. For tumor cells to efficiently metastasize, they often undergo a pernicious transformation characterized by dramatically increased migration and invasive capacity. Specifically, the spread of cancer from a localized, self-contained tumor through tissue stroma and into distant organs requires that cells achieve atypical, robust motility and the capacity to aggressively degrade extracellular matrix (ECM), enabling them to invade surrounding tissues and vessels of the blood and lymphatic systems, and subsequently establish nascent, secondary tumors [5,6]. The enhanced migration and invasive capacity of metastatic tumor cells are the subject of intense investigation, and it is now appreciated that several forms of cancer cell motility exist (i.e., single-cell, mesenchymal and amoeboid, protease-dependent, protease-independent, and collective migration) [7,8]. Consider- able progress has been made in identifying the potential molecular components that mediate cell migration, and there is growing evidence that intracellular vesicle trafficking of key proteins is crucial for efficient migration [9]. In tumor cell migration, the recycling of focal adhesion proteins (i.e., integrin receptor recycling) through endocytosis/exocytosis is now thought to contribute to the maintenance of polarized movement [10,11]. Moreover, endocytosis/exocytic trafficking has been implicated in the delivery of proteolytic enzymes - including matrix metallopro- teinases (MMPs) - to invadopodia, specialized protrusions utilized by cells for degradation of ECM [12,13]. Ferlin proteins, an evolutionarily ancient family of large integral membrane proteins [14], have been implicated in vesicle trafficking in a variety of physiological settings. All mammalian ferlins derive their names based on homology to the C. elegans protein FER-1 (FERtilization defective-1). In roundworms, fer-1 is required for the fusion of specialized vesicles (membranous organelles) with the plasma membrane at the leading edge of cell migration in spermatozoa; in the PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39766