Myoferlin (MYOF) is a member of the evolutionarily conserved ferlin family of proteins, noted for their role in a variety of membrane processes, including endocytosis, repair, and vesicular transport. Notably, ferlins are implicated in Caenorhabditis elegans sperm motility (Fer-1), mammalian skeletal muscle development and repair (MYOF and dysferlin), and presynaptic transmission in the auditory system (otoferlin). In this paper, we demonstrate that MYOF plays a previously unrecognized role in cancer cell invasion, using a combination of mathematical modeling and in vitro experiments. Using a real-time impedance-based invasion assay (xCELLigence), we have shown that lentiviral-based knockdown of MYOF significantly reduced invasion of MDA-MB-231 breast cancer cells in Matrigel bioassays. Based on these experimental data, we developed a partial differential equation model of MYOF effects on cancer cell invasion, which we used to generate mechanistic hypotheses. The mathematical model predictions revealed that matrix metalloproteinases (MMPs) may play a key role in modulating this invasive property, which was supported by experimental data using quantitative RT-PCR screens. These results suggest that MYOF may be a promising target for biomarkers or drug target for metastatic cancer diagnosis and therapy, perhaps mediated through MMPs.cancer invasion | RNAi | partial differential equation models | metastasis A majority of cancer deaths are related not to the primary tumor itself, but rather the formation of disseminated metastases (1). Cancer spread requires that cells achieve atypical motility, which enables them to invade surrounding tissues and vessels of the blood and lymphatic systems (2-4). Thus, understanding the mechanisms and signaling processes that lead to invasive cell behavior may lead to new therapeutic approaches for controlling and treating cancer.The fundamental mechanisms of invasive cancer cell movement are largely conserved across a wide range of cell types, with some of the protease dependent and protease independent movement types demonstrated by cancer cells also seen in organisms as diverse as unicellular organisms, slime molds, and white blood cells. The ferlin family is an evolutionarily ancient family of proteins (5), which are known to affect processes crucial to migration and invasion, including membrane fusion and repair, vesicle transport, endocytosis, protein recycling and stability, and cell motility (6-13). Thus, one might expect the ferlin family to be good candidates for cancer proteins, although they have not previously been investigated in this capacity. In Caenorhabditis elegans, spermatozoa exhibit amoeboid movement, and mutations in the fer-1 gene [an orthologue of myoferlin (MYOF)] result in immobility and infertility (13). In humans, MYOF has been implicated in a variety of cellular processes, including myoblast fusion, growth factor receptor stability, endocytosis, and endothelial cell membrane repair (6,8,(10)(11)(12); however until now its role in cancer cell movement ha...

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.

Cell migration plays a central role in the invasion and metastasis of tumors. As cells leave the primary tumor, they undergo an epithelial to mesenchymal transition (EMT) and migrate as single cells. Epithelial tumor cells may also migrate in a highly directional manner as a collective group in some settings. We previously discovered that myoferlin (MYOF) is overexpressed in breast cancer cells and depletion of MYOF results in a mesenchymal to epithelial transition (MET) and reduced invasion through extracellular matrix (ECM). However, the biomechanical mechanisms governing cell motility during MYOF depletion are poorly understood. We first demonstrated that lentivirus-driven shRNA-induced MYOF loss in MDA-MB-231 breast cancer cells (MDA-231MYOF-KD) leads to an epithelial morphology compared to the mesenchymal morphology observed in control (MDA- 231LTVC) and wild-type cells. Knockdown of MYOF led to significant reductions in cell migration velocity and MDA- 231MYOF-KD cells migrated directionally and collectively, while MDA-231LTVC cells exhibited single cell migration. Decreased migration velocity and collective migration were accompanied by significant changes in cell mechanics. MDA-231MYOF-KD cells exhibited a 2-fold decrease in cell stiffness, a 2-fold increase in cell-substrate adhesion and a 1.5-fold decrease in traction force generation. In vivo studies demonstrated that when immunocompromised mice were implanted with MDA- 231MYOF-KD cells, tumors were smaller and demonstrated lower tumor burden. Moreover, MDA- 231MYOF-KD tumors were highly circularized and did not invade locally into the adventia in contrast to MDA- 231LTVC-injected animals. Thus MYOF loss is associated with a change in tumor formation in xenografts and leads to smaller, less invasive tumors. These data indicate that MYOF, a previously unrecognized protein in cancer, is involved in MDA-MB-231 cell migration and contributes to biomechanical alterations. Our results indicate that changes in biomechanical properties following loss of this protein may be an effective way to alter the invasive capacity of cancer cells.

A majority of the studies examining the molecular regulation of human labor have been conducted using single gene approaches. While the technology to produce multi-dimensional datasets is readily available, the means for facile analysis of such data are limited. The objective of this study was to develop a systems approach to infer regulatory mechanisms governing global gene expression in cytokine-challenged cells in vitro, and to apply these methods to predict gene regulatory networks (GRNs) in intrauterine tissues during term parturition. To this end, microarray analysis was applied to human amnion mesenchymal cells (AMCs) stimulated with interleukin-1β, and differentially expressed transcripts were subjected to hierarchical clustering, temporal expression profiling, and motif enrichment analysis, from which a GRN was constructed. These methods were then applied to fetal membrane specimens collected in the absence or presence of spontaneous term labor. Analysis of cytokine-responsive genes in AMCs revealed a sterile immune response signature, with promoters enriched in response elements for several inflammation-associated transcription factors. In comparison to the fetal membrane dataset, there were 34 genes commonly upregulated, many of which were part of an acute inflammation gene expression signature. Binding motifs for nuclear factor-κB were prominent in the gene interaction and regulatory networks for both datasets; however, we found little evidence to support the utilization of pathogen-associated molecular pattern (PAMP) signaling. The tissue specimens were also enriched for transcripts governed by hypoxia-inducible factor. The approach presented here provides an uncomplicated means to infer global relationships among gene clusters involved in cellular responses to labor-associated signals.

Tissues are composed of multiple cell types in a well-organized three-dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue-engineered constructs should have well-defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two-dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well-defined microstructure were constructed into a multilayer cell-scaffold complex using low pressure carbon dioxide (CO(2)) and nitrogen (N(2)). The mouse ES cells in the assembled constructs were viable, retained the ES cell-specific gene expression of Oct-4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO(2) was replaced with N(2). The compressed gas-assisted bioassembly of stem cell-polymer constructs opens up a new avenue for tissue engineering and cell therapy.

Introduction: Ferlins are a family of proteins that play a variety of roles associated with plasma membrane dynamics in eukaryotes, including plasma membrane repair, regulation of membrane expression of receptors, and endocytosis. Dysferlin was recently reported to regulate platelet endothelial cellular adhesion molecule-1 (PECAM-1) in endothelial cells (1). We have shown that ablation of myoferlin (MYOF) in breast tumor cells attenuates invasion in vitro (in review, J Cell Sci). Moreover, loss of MYOF was found to be associated with reduced phosphorylation of a number of receptor tyrosine kinases (in press, PNAS), and others have reported on the role of MYOF in tyrosine kinase receptors expression at the plasma membrane (2). These results prompted us to hypothesize that MYOF may be involved in cell-matrix adhesion. Methods & Results: We used lentiviral-mediated RNA interference (RNAi) gene silencing to generate stable MYOF-deficient MDA-MB-231 cells and confirmed MYOF reduction with immunoblotting and qRT-PCR. RNAi control cells were generated in tandem with a non-human gene targeting construct. Relative adhesion and spreading of the wild-type, control, and MYOF-deficient epithelial cells were evaluated using a real-time electrical impedance measurement system (xCELLigence, Roche Applied Science). Results show a significant increase in the adhesion and spreading of the MYOF-deficient cells in comparision to wild-type and control cells. A trypsin assay developed using the same electrical impedance system demonstrated greater adhesion strength in the MYOF-deficient cells. This was measured by the duration of time necessary to reduce the pre-trypsin-treatment impedance by one-half. These results were further validated using a conventional parallel plate device, in which MYOF-deficient cells were found to be more adherent than controls when subjected to a range of shear stresses (1-10 dynes/cm2). PCR array screening of extracellular matrix related genes (SABiosciences) in MYOF-deficient and RNAi control MDA-MB-231 cells indicated that intercellular adhesion molecule 1 (ICAM-1/CD54) and vitronectin mRNA levels are increased following MYOF-diminution, and may potentially contribute to the mechanism behind the altered adhesion in MYOF-deficient cells. Conclusion: Myoferlin is important for the regulation of breast cancer epithelial cell adhesion. Since dysregulation of adhesion is often associated with cancer invasion and metastasis, myoferlin may play a role in these processes in breast carcinoma. (1) Sharma A, Yu C, Leung C, Trane A, Lau M, Utokaparch S, et al. A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis. Arterioscler Thromb Vasc Biol 2010 Nov;30 (11):2196-204. (2) Yu C, Sharma A, Trane A, Utokaparch S, Leung C, Bernatchez P. Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo. Vascul Pharmacol 2011 May 6. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5180. doi:1538-7445.AM2012-5180

Introduction: Ferlins are an evolutionarily conserved family of proteins that play a variety of roles associated with plasma membrane dynamics in eukaryotes, including membrane repair and endocytosis. Myoferlin (MYOF) is a mammalian ferlin protein with homology to ancestral Fer-1, a C. elegans protein that regulates Ca2+-dependent spermatic membrane fusion, a key mechanism underlying the amoeboid-like movements of C. elegans sperm. Consequently, mutations in fer-1 lead to immotile C. elegans sperm and infertility. This immotility phenotype in C. elegans spermatids is of interest since metastatic cancer cells are often associated with an amoeboid appearance as they invade surrounding tissues and blood and lymphatic vessels. Given this, we hypothesized that MYOF plays a role in cancer cell biology by contributing to the stability of the plasma membrane during proliferation, migration and/or invasion. Methods and Results: We first demonstrated the expression of MYOF protein and mRNA in the malignant, invasive breast cell line MDA-MB-231 using immunoblotting and real time polymerase chain reaction (RT-PCR) methods, respectively. Using lentiviral-mediated RNA interference (RNAi) gene silencing technology, we then generated stable MYOF-deficient MDA-MB-231 (231) cells and confirmed MYOF reduction with immunoblotting. RNAi control cells were generated in tandem with a non-human gene targeting construct. Interestingly, when MYOF-deficient 231 cells were grown in culture, a reversion from a mesenchymal to a more epithelial morphology was observed, and sustained through subsequent passages. This suggests that MYOF may be critical for one or more events during epithelial-mesenchymal transition (EMT). Using a commercial colorimetric assay for the quantification of viable cells, we did not detect any significant effect of MYOF deficiency on the proliferation of the 231 cells. Using the Boyden Chamber assay, MYOF-deficient 231 cells show a reduced invasive capacity through Matrigel compared to RNAi control cells, while no difference was observed for the migration capability of MYOF-deficient 231 cells. The invasive potential of 231 cells was further evaluated with a real-time invasion assay (xCELLigence, Roche Applied Science) that also showed a reduction in the invasive potential of the MYOF-deficient cells. These results suggest that MYOF may be important for cancer cell invasion. PCR array screening of extracellular matrix related genes (SABiosciences) in MYOF-deficient and RNAi control 231 cells indicates that matrix metalloproteinase levels may be affected by MYOF-diminution, and thereby contribute to the mechanism behind the altered invasion of MYOF-deficient cells. Conclusion: Myoferlin may play a role in cancer progression by promoting cellular invasion, and therefore be a potentially important prospective target for cancer therapy and a marker for prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2347. doi:10.1158/1538-7445.AM2011-2347

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