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.
Highlights
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]
In a survey using a Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)-based array of epithelial-mesenchymal transition (EMT) markers, we found that the mRNA levels appeared to correlate with the Western results in that fibronection mRNA was down-regulated 3.46-fold while that of E-cadherin was upregulated 2.58-fold in 231MYOF-KD cells compared with controls, no significant change was found for vimentin (Table S1)
We provide in silico and experimental evidence that MYOF plays a critical and previously unrecognized role in breast tumor cell invasion
Summary
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]. For tumor cells to efficiently metastasize, they often undergo a pernicious transformation characterized by dramatically increased migration and invasive capacity. 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 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]. Endocytosis/exocytic trafficking has been implicated in the delivery of proteolytic enzymes - including matrix metalloproteinases (MMPs) - to invadopodia, specialized protrusions utilized by cells for degradation of ECM [12,13]
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