A conserved region within interferon regulatory factor 5 controls breast cancer cell migration through a cytoplasmic and transcription-independent mechanism.

BACKGROUND: Migration 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. RESULTS: A 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. CONCLUSIONS: These 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.

IRF5 is a novel regulator of CXCL13 expression in breast cancer that regulates CXCR5(+) B- and T-cell trafficking to tumor-conditioned media.

Clinical studies using prognostic and predictive signatures have shown that an immune signal emanating from whole tumors reflects the level of immune cell infiltration--a high immune signal linked to improved outcome. Factors regulating immune cell trafficking to the tumor, however, are not known. Previous work has shown that expression of interferon regulatory factor 5 (IRF5), a critical immune regulator, is lost in ~80% of invasive ductal carcinomas examined. We postulated that IRF5-positive and -negative breast tumors would differentially regulate immune cell trafficking to the tumor. Using a focused tumor inflammatory array, differences in cytokine and chemokine expression were examined between IRF5-positive and -negative MDA-MB-231 cells grown in three-dimensional culture. A number of cytokines/chemokines were found to be dysregulated between cultures. CXCL13 was identified as a direct target of IRF5 resulting in the enhanced recruitment of B and T cells to IRF5-positive tumor-conditioned media. The ability of IRF5 to regulate mediators of cell migration was confirmed by enzyme-linked immunosorbent assay, chromatin immunoprecipitation assay, small interfering RNA knockdown and immunofluorescence staining of human breast tumor tissues. Analysis of primary immune cell subsets revealed that IRF5 specifically recruits CXCR5(+) B and T cells to the tumor; CXCR5 is the receptor for CXCL13. Analysis of primary breast tumor tissues revealed a significant correlation between IRF5 and CXCL13 expression providing clinical relevance to the study. Together, these data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may, in combination with CXCL13, serve as a novel prognostic marker for antitumor immunity.

Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis.

INTRODUCTION: New signaling pathways of the interleukin (IL) family, interferons (IFN) and interferon regulatory factors (IRF) have recently been found within tumor microenvironments and in metastatic sites. Some of these cytokines stimulate while others inhibit breast cancer proliferation and/or invasion. IRFs, a family of nine mammalian transcription factors, have multiple biologic functions that when dysregulated may contribute to tumorigenesis; most well-known are their roles in regulating/initiating host immunity. Some IRF family members have been implicated in tumorigenesis yet little is still known of their expression in primary human tumors or their role(s) in disease development/progression. IRF5 is one of the newer family members to be studied and has been shown to be a critical mediator of host immunity and the cellular response to DNA damage. Here, we examined the expression of IRF5 in primary breast tissue and determined how loss of expression may contribute to breast cancer development and/or progression. METHODS: Formalin-fixed paraffin-embedded archival breast tissue specimens from patients with atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) were examined for their expression of IRF1 and IRF5. Knockdown or overexpression of IRF5 in MCF-10A, MCF-7 and MDA-MB-231 mammary epithelial cell lines was used to examine the role of IRF5 in growth inhibition, invasion and tumorigenesis. RESULTS: Analysis of IRF expression in human breast tissues revealed the unique down-regulation of IRF5 in patients with different grades of DCIS and IDC as compared to IRF1; loss of IRF5 preceded that of IRF1 and correlated with increased invasiveness. Overexpression of IRF5 in breast cancer cells inhibited in vitro and in vivo cell growth and sensitized them to DNA damage. Complementary experiments with IRF5 siRNAs made normal mammary epithelial cells resistant to DNA damage. By 3-D culture, IRF5 overexpression reverted MDA-MB-231 to normal acini-like structures; cells overexpressing IRF5 had decreased CXCR4 expression and were insensitive to SDF-1/CXCL12-induced migration. These findings were confirmed by CXCR4 promoter reporter assays. CONCLUSIONS: IRF5 is an important tumor suppressor that regulates multiple cellular processes involved in the conversion of normal mammary epithelial cells to tumor epithelial cells with metastatic potential.