absence of functional FER-1 protein, the normal amoeboid locomotion of sperm is impaired, and infertility results [15]. Human patients harboring FER1L1 (FER-1-like 1, dysferlin) mutations manifest one of two autosomal recessive forms of muscular dystrophy - limb girdle muscular dystrophy type 2B and Myoshi myopathy [16], due to the inability of skeletal muscle fiber sarcolemma to repair damaged muscle cells during the normal course of biomechanical wear and tear [17–19]. Mutations in FER1L2 (otoferlin) result in non-syndromic deafness (DFNB9), due to the failure of synaptic vesicles to fuse and exocytose their cargo at the presynaptic plasma membrane [20,21]. FER1L3 (myoferlin, MYOF), historically considered to be a muscle-specific protein, has not yet been directly associated with a distinct mammalian disorder, but recent studies have indicated that its deletion results in impaired mouse myoblast fusion into mature skeletal myotubes [22]. Furthermore, MYOF has been shown to mediate caveolae- dependent endocytosis in human endothelial cells [23]. Recent reports indicate that MYOF is critically involved in the function and/or stability of plasma membrane receptor tyrosine kinases (RTKs). Bernatchez et al. demonstrated that ablation of MYOF in vascular endothelial cells resulted in instability and rapid degradation of the vascular endothelial growth factor receptor 2 (VEGFR-2) [24]. Further research demonstrated that the expression of another angiogenic tyrosine kinase receptor, Tie- 2, is also attenuated upon MYOF depletion in endothelial cells [25]. Finally, Demonbreun et al. reported that knockout of MYOF in mouse muscle resulted in diminished insulin-growth factor-1 receptor (IGFR) response and accumulation of the receptors in vesicles targeted for degradation [26]. The studies reported above when considered in toto suggest that MYOF may be a regulator of vesicle fusion events that deliver essential cargos (e.g., growth factor receptors, cell adhesion molecules, and other cell-surface proteins) to and/or from the plasma membrane. Inasmuch as tumor cell metastatic dissemination involves the convergence of receptors, signal transduction pathways, adhesion and matrix proteolytic mole- cules at the leading edge of a tumor cell, we proposed that MYOF (and perhaps other ferlin proteins) may be involved in one or more steps in tumor progression and/or spread. Microarray and proteomic studies in the cancer literature have reported MYOF expression in breast cancer specimens and relevant cell lines [27–29]. For example, in one microarray study, MYOF was 1 of 39 genes found to be over-represented in breast carcinoma [28]. These observations prompted us to hypothesize that MYOF may be an important protein in breast cancer cells for their mobilization during cellular migration and/or invasion. We recently reported on the mathematical modeling of the role of MYOF in breast cancer cell invasion [30]. In the current manuscript, we present novel data confirming the computational modeling and show, in addition, that MYOF- deficient MDA-MB-231 human breast cancer cells exhibit a more epithelial shape compared to the mesenchymal morphol- ogy of wild-type MDA-MB-231 cells, suggesting that some features of a mesenchymal-to-epithelial transition (MET) phe- notype [31] may exist in the absence of functional MYOF protein. Matrigel and collagen I based invasion assays demon- strated that depletion of MYOF diminished the invasive ability of cancer cells. In congruence with the mathematical modeling results, we report that the decreased invasive capacity is due, at least in part, to the down-regulation of MMP expression in MYOF-deficient cells. Myoferlin in Breast Cancer EMT & Invasion Results Myoferlin in Breast Cancer Cells Correlates with Cell Invasiveness To correlate the expression of MYOF with the invasive capacity of breast cancer cells, we analyzed five mammary cell lines: MCF- 10A, MCF-7, T47D, BT549, and MBA-MB-231. The MCF-10A line was chosen as a model for non-cancerous, non-invasive mammary epithelial [32]. MCF-7 and T47D cells model non2/ low-invasive breast cancers, while BT549 and MDA-MB-231 have high invasive capacity [33]. Examination of MYOF mRNA expression in each of the cell lines indicated that MYOF expression was 2.44-fold higher in the invasive lines (BT549 and MDA-MB-231) relative to non2/low- invasive cells (MCF-10A, MCF-7, and T47D) (Figure 1A and B). In congruence, higher MYOF protein expression was observed by immunoblotting in the highly-invasive breast cancer cell lines relative to those with low invasive potential (Figure 1C). To expand upon this, we examined the transcriptional profiling data available on 51 established breast cancer lines generated by Neve and colleagues (http://cancer.lbl.gov/breastcancer/data) [34]. Those 14 cell lines which clustered into the invasive, basal B-like subset exhibited statistically significantly greater MYOF expression compared to the 25 cell lines of the luminal phenotype as determined by two MYOF probesets (201798_s_at and 211864_s_at, Figure S1). Building on these observations, we mined published microarray data available through the ArrayExpress database (www.ebi.ac. uk/arrayexpress) [35]. Our query returned microarray data (accession: E-TABM-276) from Cheng et al. [36] consisting of a panel of 23 microdissected primary invasive ductal carcinoma tissues, 28 adjacent stromal tissues, and 10 healthy controls. When MYOF expression was categorized by disease state, we observed significantly higher MYOF mRNA expression in tissues from patients with invasive ductal carcinoma compared with samples from healthy individuals (Figure S2). Collectively, these data suggest a potential correlation between MYOF over-expression and invasive breast carcinoma. Myoferlin Knockdown Promotes a Mesenchymal to Epithelial Change Given a possible association between MYOF expression and invasive potential, we examined whether MYOF contributed to breast cancer cell invasion. We selected the MDA-MB-231 cells for these experiments since they exhibited high levels of MYOF protein expression in our survey, and were previously shown to have the greatest invasive capacity compared to a large panel of breast cancer cell lines [34]. Stable lines of MYOF-deficient MDA-MB-231 cells (231MYOF-KD) were generated using lentivi- rus-based delivery of short hairpin ribonucleic acids (shRNAs) targeting human MYOF (Figure S3). Control cell lines (231LTV-ctrl) were generated in parallel using a non-human gene targeting construct. Selective knockdown of MYOF protein expression was confirmed by immunoblotting (Figure 1D). Of the three constructs initially screened (#10628, 10630, and 1522), #10628 yielded the most efficient knockdown (mean 94% compared with controls, n = 4) and was used in subsequent experiments. An unexpected, but interesting shape change appeared in the 231MYOF-KD cells, wherein they exhibited a more epithelial-like (cobblestone) morphology which diverged from the fibroblastic, spindle shape of the wild type and 231LTV-ctrl cells (Figures 2 and 3). The 231MYOF-KD morphology was stable, and was maintained after 10 continual passages in culture (data not shown). By immunofluorescent microscopy, we noted that 231MYOF-KD cells PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39766