Abstract
BackgroundThe membrane cytoskeletal crosslinker, ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells. Our group previously showed that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in deregulation of cell-cell contacts and scattering of epithelial cells. In particular, ezrin phosphorylation on Y477 by Src is specific to ezrin within the ERM family, and is required for HGF-induced scattering of epithelial cells. We therefore sought to examine the role of Y477 phosphorylation in ezrin on tumor progression.MethodsUsing a highly metastatic mouse mammary carcinoma cell line (AC2M2), we tested the effect of over-expressing a non-phosphorylatable form of ezrin (Y477F) on invasive colony growth in 3-dimensional Matrigel cultures, and on local invasion and metastasis in an orthotopic engraftment model.ResultsAC2M2 cells over-expressing Y477F ezrin exhibited delayed migration in vitro, and cohesive round colonies in 3-dimensional Matrigel cultures, compared to control cells that formed invasive colonies with branching chains of cells and numerous actin-rich protrusions. Moreover, over-expression of Y477F ezrin inhibits local tumor invasion in vivo. Whereas orthotopically injected wild type AC2M2 tumor cells were found to infiltrate into the abdominal wall and visceral organs within three weeks, tumors expressing Y477F ezrin remained circumscribed, with little invasion into the surrounding stroma and abdominal wall. Additionally, Y477F ezrin reduces the number of lung metastatic lesions.ConclusionsOur study implicates a role of Y477 ezrin, which is phosphorylated by Src, in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic/predictive marker or treatment target for invasive human breast cancer.
Highlights
The membrane cytoskeletal crosslinker, ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells
Using a model of orthotopic engraftment of a mouse mammary carcinoma tumor cell line (AC2M2) [20], we found that over-expression of a nonphosphorylatable Y477F ezrin mutant markedly decreased local invasion of primary tumor transplants, compared to control vector expressing tumor transplants which rapidly infiltrated into underlying abdominal wall and visceral tissues
Expression of Y477F ezrin mutant attenuates migration of breast carcinoma cells Since cell migration is an important early step in tumor invasion, we examined the role of Y477 ezrin on migration function of a highly metastatic carcinoma variant cell line, AC2M2, derived from the mouse mammary tumor cell line, SP1 [35]
Summary
The membrane cytoskeletal crosslinker, ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells. Ezrin phosphorylation on Y477 by Src is specific to ezrin within the ERM family, and is required for HGF-induced scattering of epithelial cells. Gene [9,10] and protein [11,12,13,14,15,16,17] expression profiling have revealed a marked increase in ezrin expression in a variety of human and rodent cancers compared to nonmalignant tissue counterparts. Increased cytoplasmic expression of ezrin is frequently associated with dedifferentiation, invasiveness, and poor prognosis in human breast cancers; compared to membranous apical expression in non-malignant epithelial tissues [18]. Ectopic expression of the suppressor gene, RhoBTB2, causes dephosphorylation of ezrin and inhibits migration and metastasis of breast carcinoma cells [23]
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