Myoferlin in Breast Cancer EMT & Invasion Figure 5. Myoferlin depletion reduces invasive but not migratory capacity of MDA-MB-231s control (LTV-ctrl) and MYOF depleted cells (MYOF-KD). (A) Boyden chamber migration assay of MDA-MB-231 cells moving across 8 mm porous membranes towards a 10% serum gradient for 24 h (mean 6 s.d., n = 3, unpaired 2-tailed t-test). (B) 24 h Boyden chamber invasion results of MDA-MB-231 cells across a 100% Matrigel coated 8 mm porous membrane towards a 10% serum chemoattractant (mean 6 s.d., n = 3, unpaired 2-tailed t-test). doi:10.1371/journal.pone.0039766.g005 Cp value ,30), only MMP1 was found to be significantly regulated ($ 62 fold-change) at the transcription level by MYOF- depletion. Discussion We provide in silico and experimental evidence that MYOF plays a critical and previously unrecognized role in breast tumor cell invasion. Mining of transcriptome and proteome databases from breast cancer cell lines revealed higher levels of MYOF mRNA and protein in the more invasive lines compared with poorly invasive breast tumor cells or normal mammary epithelial cells (i.e., MCF-10A). Published microarray datasets using breast cancer cell lines [34] also support greater MYOF expression in the invasive basal B group compared with the luminal subtype. In a separate study, MYOF was found to be 1 of 39 genes selectively over-expressed in breast carcinoma using 20 human breast cancer cases [28]. These data suggested to us that MYOF is a potentially important protein in breast cancer biology. The premise that MYOF contributes to cancer invasion was based on several reports implicating a role for ferlin proteins in intracellular vesicle trafficking, including cell motility. For example, Ward and colleagues demonstrated that temperature- sensitive mutations in the ancestral fer-1 gene in C. elegans led to fertility impairments yielding immotile spermatozoa secondary to defects in vesicle recycling at the leading edge of the cell [15,44]. Mechanistically, mutations in C2 domains of fer-1 altered calcium sensitivity and subsequently impeded calcium-dependent fusion of intracellular vesicles to the plasma membrane that drives the amoeboid movement of the roundworm sperm cells [15]. Vesicle trafficking has been implicated in several important steps during cell motility [9]. Based on these observations, coupled with the observation that MYOF expression was increased in breast cancer specimens and breast cancer cell lines with high invasive potential, we hypothesized that MYOF depletion would impair the motility of breast cancer cells. And this appeared to be the case. End-point Boyden chamber assays accessing cell motility initially detected no overall change in migration during a 24 hour assay period. This was somewhat surprising in light of the work of Achanzar et al. [45] demonstrating that mutation of fer-1 resulted in profound defects in amoeboid migration of C. elegans sperm cells. However, recent work from our lab using live-cell imaging clearly shows that MYOF depletion leads to fundamental changes in the mode of breast tumor cell motility (Volakis et al., in preparation, J Biomechanics), suggesting that MYOF loss also contributes to disturbances in cell migration on two-dimensional surfaces (work in progress). Tumor cell invasion into distant tissues to establish secondary malignancies is a hallmark of cancer metastasis and cancer-related death [4]. In many cases, enhanced tumor cell migration as well as local and distant invasion are preceded by an EMT that converts sessile epithelial cells into actively migratory and invasive cells [46,47]. In order for efficient migration and invasion to occur, in many instances cancer cells must secrete several MMPs for basement membrane and stromal ECM degradation [48]. We probed the invasive capacity of breast tumor cells (MDA-MB-231) following shRNA directed depletion of MYOF (shRNAMYOF), and noted a profound deficit in the ability of cells to invade through Matrigel (basement membrane mimic) [49] and collagen type I (stromal ECM mimic) [50]. These observations indicated that MYOF disruption may perturb one or more MMPs that are essential for effective cell invasion through the ECM or collagen I [48]. Indeed, screens of several MMPs using qRT-PCR and protein immunoassay arrays suggested that MMPs-1, 3, 8, 12, 13, 14, and 16 were down-regulated in MDA-MB-231 cells in which MYOF was knocked down by RNAi (Figure S5 and Table S2). Interestingly, MMP-9 appeared to be up-regulated under these same conditions, indicating the MYOF depletion results in selective changes in MMPs. To evaluate the effect of abolishing MYOF on MMP expression, we probed MMP-1 further, because our data revealed a nearly complete loss of MMP-1 when shRNAMYOF was expressed in MDA-MB-231 cells. Quantitative RT-PCR and Western blotting showed essentially no detectable MMP-1 mRNA or protein in 231MYOF-KD cells. Moreover, we detected virtually no pro-MMP-1 reactive protein by ELISA. Thus, it appears that MYOF may play a role in MMP gene expression in addition to other mechanisms it may influence, such as MMP secretion via vesicle trafficking. We are currently exploring the mechanistic basis for this apparent alteration in MMP gene expression in human breast tumor cells. To authenticate that our shRNA results were not due to off- target effects on the MMP mRNAs, we conducted an extensive in silico analysis using several software packages available in the PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39766