Abstract
BackgroundMigration of breast cancer cells out of a duct or lobule is a prerequisite for invasion and metastasis. However, the factors controlling breast cancer cell migration are not fully elucidated. We previously found that expression of the transcription factor interferon regulatory factor 5 (IRF5) is significantly decreased as a breast lesion progresses from a non-malignant stage to ductal carcinoma in situ and is eventually lost in ~80% of invasive ductal carcinomas examined. Human in vitro and murine in vivo models of invasive breast cancer confirmed an important role for IRF5 in regulating cell motility, invasion and/or metastasis; yet, the mechanism(s) by which this occurs is not known. Since IRF5 is primarily expressed in the cytoplasm of human mammary epithelial cells, we hypothesized that IRF5 may function in a transcription-independent manner to control intrinsic cell migration.ResultsA series of IRF5 deletion mutants were tested in cell motility, invasion and migration assays. A novel, conserved 10 amino acid domain was identified that regulates mammary epithelial cell migration. This region (∆115-125) is downstream of IRF5′s DNA binding domain and therefore when absent, retains IRF5 transcription activity but loses cell migration control. An IRF5 construct with a mutated nuclear localization signal further confirmed that IRF5 controls migration in a cytoplasmic and transcription-independent manner. Candidate cytoskeletal molecules were identified in MDA-MB-231 cells to interact with IRF5 by immunoprecipitation and mass spectrometry analysis. α6-tubulin was independently confirmed to interact with endogenous IRF5 in MCF-10A cells. Alterations in F-actin bundling after staining EV- and IRF5-231 cells with phalloidin suggests that IRF5 may control cell migration/motility through its interaction with cytoskeletal molecules that contribute to the formation of F-actin networks. Last and most notably, we found that IRF5′s control of cell migration is not restricted to mammary epithelial cells but functions in other epithelial cell types suggesting a more global role for this newly identified cell migratory function of IRF5.ConclusionsThese findings are significant as they identify a new regulator of epithelial cell migration and provide specific insight into the mechanism(s) by which loss of IRF5 expression in mammary epithelial cells contributes to breast cancer metastasis.
Highlights
Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors that are expressed in vertebrates
interferon regulatory factor 5 (IRF5) expression inhibits the migration of breast cancer cells Experimental in vitro and in vivo data support a role for IRF5 in mammary epithelial cell migration and metastasis
Given that all three of these cytoskeletal molecules have been implicated in cancer migration, invasion and metastasis formation [33], in part, through their ability to participate in filamentous (F)-actin networking that is integral for cell motility [33,34], we examined F-actin networking in EV-231 and IRF5-231 cells and found distinct and condensed F-actin bundling at the nucleus of IRF5-231 cells as compared with EV-231 cells (Figure 6B)
Summary
Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors that are expressed in vertebrates. Most well known is the induction of IRF5 activation by MyD88-dependent Toll like receptor (TLR) signaling [7,12,13,14] In this pathway, ligand bound to receptor induces a cascade of events whereby IRF5 binds to MyD88 and TRAF6 [3,5,13,14], undergoes several post-translational modifications including phosphorylation and possibly ubiquitination [10], and forms hetero- or homodimers that translocate to the nucleus [4,6,12,13,15,16]. IRF5 has been shown to positively regulate cytokines such as Type I interferons (IFNs), interleukin (IL)-6, IL-12, IL-1β, and TNF-α while suppressing IL-10 expression [11,14] Most studies of this protein to date have been performed in lymphocytes, IRF5 is classically thought of as a transcriptional immune regulator [4,7,11,15]. Since IRF5 is primarily expressed in the cytoplasm of human mammary epithelial cells, we hypothesized that IRF5 may function in a transcription-independent manner to control intrinsic cell migration
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