Interferon regulatory factor-1 regulates reconstituted extracellular matrix (rECM)-mediated apoptosis in human mammary epithelial cells

Oncogene (2007) 26, 2017–2026 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Interferon regulatory factor-1 regulates reconstituted extracellular matrix (rECM)-mediated apoptosis in human mammary epithelial cells ML Bowie1, MM Troch1, J Delrow2, EC Dietze1, GR Bean1, C Ibarra1, G Pandiyan3 and VL Seewaldt1,3 1 Department of Medicine, Duke University, Durham, NC, USA; 2Department of Genomics/Shared Resources, Fred Hutchinson Cancer Research Center, Seattle, WA, USA and 3Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA Interactions between extracellular matrix (ECM) and mammary epithelial cells are critical for mammary gland homeostasis and apoptotic signaling. Interferon regulatory factor-1 (IRF-1) is a transcriptional regulator that promotes apoptosis during mammary gland involution and p53-independent apoptosis. We have recently shown that rapid cell surface tamoxifen (Tam) signaling promotes apoptosis in normal human mammary epithelial cells that were acutely damaged by expression of human papillomavirus type-16 E6 protein (*HMEC-E6). Apoptosis was mediated by recruitment of CREB-binding protein (CBP) to the c-activating sequence (GAS) element of the IRF-1 promoter, induction of IRF-1 and caspase-1/-3 activation. Here, we show that growth factor-depleted, reconstituted ECM (rECM), similar to Tam, promotes apoptosis in *HMEC-E6 cells through induction of IRF-1. Apoptosis was temporally associated with recruitment of CBP to the GAS element of the IRF-1 promoter, induction of IRF-1 expression and caspase-1/-3 activation. Small interfering RNA-mediated suppression of IRF-1 protein expression in *HMEC-E6 cells blocked (1) induction of IRF-1, (2) caspase-1/-3 activation and (3) apoptosis. These observations demonstrate that IRF-1 promotes rECM-mediated apoptosis and provide evidence that both rECM and rapid Tam signaling transcriptionally activate IRF-1 through recruitment of CBP to the IRF-1 GAS promoter complex. Oncogene (2007) 26, 2017–2026. doi:10.1038/sj.onc.1210013; published online 2 October 2006 Keywords: mammary epithelial cells; IRF-1; apoptosis; extracellular matrix; tamoxifen Introduction Interferon regulatory factor-1 (IRF-1) was originally identified as a DNA-binding factor on the mouse interferon beta (IFN-b) promoter and is known to play Correspondence: Dr VL Seewaldt, Duke University, Box 2628, MSRB, Durham, NC 27710, USA. E-mail: Received 15 March 2006; revised 1 August 2006; accepted 4 August 2006; published online 2 October 2006 an important role in promoting apoptosis in response to viral infections (Miyamoto et al., 1988). IRF-1 can be induced by type II-IFN (IFN-g) (Romeo et al., 2002) and is known to participate in both p53-dependent and -independent apoptotic signaling (Tamura et al., 1995; Tanaka et al., 1996). Recently, IRF-1 was shown to participate in both type IFN-g and anti-estrogen ICI 182 780 p53-independent apoptotic signaling in human breast cancer cell lines (Bouker et al., 2004; Porta et al., 2005). Taken together, these observations suggest that IRF-1 plays a role in p53-independent apoptotic signaling in mammary epithelial cells. There is also growing evidence that IRF-1 plays an important role in mammary gland homeostasis and hormone responsiveness. IRF-1 has been implicated to be involved in ICI 182 780 anti-estrogen resistance in breast cancer (Gu et al., 2002). IRF-1 is induced by the estrogen agonist/antagonist, tamoxifen (Tam) and the pure estrogen antagonist, ICI 182 780 (Bouker et al., 2004; Bowie et al., 2004). Taken together, these observations support the role of IRF-1 signaling in mammary gland homeostasis and estrogen/anti-estrogen signaling. The ‘classic’ or genomic mechanism of 17-b-estradiol (E2) action requires the presence of the estrogen receptor (ER), the E2/ER complex binding to an estrogen response element and changes in both transcription and translation. However, recent evidence suggests that estrogen, and anti-estrogens, may also act through rapid, ‘non-classic’ signaling pathways in human mammary epithelial cells (HMECs) (Kelly and Levin, 2001; Marquez and Pietras, 2001; Dietze et al., 2004). Unlike ER( þ ) human breast cancers, HMECs typically express low nuclear levels of ER (ER-‘poor’). Although HMECs are ER-‘poor’, unlike ER() breast cancer cells, HMECs are not Tam-resistant. We recently demonstrated that although therapeutic levels of Tam (1.0 mM) promoted growth arrest in HMEC controls, equimolar concentrations of Tam induced apoptosis in ER-‘poor’ *HMEC-E6 through a rapid, ‘non-classic’ signaling pathway (Dietze et al., 2001, 2004; Bowie et al., 2004). Tam induced apoptosis in *HMEC-E6 cells through (1) rapid cell-surface-mediated modulation of AKT phosphorylation, (2) recruitment of STAT1 and the coactivator, CREB-binding protein (CBP), to the IRF-1 regulates ECM-mediated apoptosis ML Bowie et al 2018 g-activating sequence (GAS) element of the IRF-1 promoter and (3) induction of IRF-1 (Bowie et al., 2004; Dietze et al., 2004). These observations suggest a role for IRF-1 in regulating rapid Tam signaling and promoting p53-independent apoptosis in *HMEC-E6 cells. Carcinogenesis is thought to be a multistep process resulting from the accumulation of genetic damage. However, not all damaged mammary epithelial cells progress to become invasive breast cancers and, instead, are thought to be eliminated by apoptosis. Breast tissue is composed of mammary epithelial cells that rest on extracellular matrix (ECM). Interactions between epithelial cells and ECM regulate mammary gland homeostasis by promoting a coordinated balance between proliferation and apoptosis (Folkman and Moscona, 1978; Petersen et al., 1992; Farrelly et al., 1999; Stupack and Cheresh, 2002). Laminins are heterotrimeric ECM glycoproteins that mediate many of the regulatory functions of ECM (Aberdam et al., 2000). Laminin-5 (a3A, b3 and g2) is the most abundant ECM glycoprotein produced by mammary epithelial cells (D’Ardenne et al., 1991). Our prior studies showed that although contact with reconstituted ECM (rECM) promoted growth arrest in HMEC controls, contact between rECM and *HMECE6 cells promotes p53-independent apoptosis through a laminin-5/a3b1-integrin signaling pathway; interruption of laminin-5/a3b1-integrin signaling blocked apoptosis (Seewaldt et al., 2001; Dietze et al, 2005). Given the observations that IRF-1 is important for response to acute cellular damage and p53-independent apoptotic signaling in mammary epithelial cells and our recent observation that Tam promotes p53-independent apoptosis in *HMEC-E6 cells through induction of IRF-1 (Bowie et al., 2004; Dietze et al., 2004), we hypothesized that rECM may also promote p53-independent apoptosis in *HMEC-E6 cells through induction of IRF-1. Here, we show that rECM, similar to Tam, promotes recruitment of STAT1 and CBP to the GAS element of the IRF-1 promoter and subsequent (...truncated)