IRF-1 expression induces apoptosis and inhibits tumor growth in mouse mammary cancer cells in vitro and in vivo

Abstract

Interferon regulatory factor-1 (IRF-1) is a nuclear transcription factor that mediates interferon and other cytokine effects and appears to have antitumor activity in vitro and in vivo in cancer cells. We have constructed a recombinant adenoviral vector (Ad-IRF-1) that infects mammary cells with high efficiency and results in high levels of functional IRF-1 protein in transfected cells. Overexpression of IRF-1 in two mouse breast cancer cell lines, C3-L5 and TS/A, resulted in apoptosis in these cell lines as assessed by Annexin V staining. The involvement of caspases was confirmed by significant inhibition of apoptosis by a caspase inhibitor, and by demonstration of caspase-3 activity, cleavage of caspase-3, and PARP cleavage. Interestingly, the growth of nonmalignant breast cell lines C127I and NMuMG did not appear to be inhibited by IRF-1 overexpression. Suppression of growth for breast cancer cell lines in vivo was demonstrated by both preinfection of breast cancer cells ex vivo and by intratumoral injection of Ad-IRF-1 into established tumors in their natural hosts. The mechanism of apoptosis may involve the transcriptional upregulation of bak, caspase-8, and caspase-7 expression. These data support the antitumor potential of IRF-1 and the use of agents that increase IRF-1 in breast cancer.

Interferon regulatory factor-1-induced apoptosis mediated by a ligand-independent fas-associated death domain pathway in breast cancer cells

“…We established that adenoviral transgene expression of IRF-1 induces a similar caspase-mediated apoptosis, and since IRF-1 is known to mediate both physiologic and pharmacologic apoptosis we utilized Ad-IRF-1 to facilitate our studies. IRF-1 expression correlated with an apoptotic time course of 24-36 h and this is supported by a matching time-course immunoblot of caspase-8, -3 and -7 cleavages, as well as current literature that associates IRF-1 and caspase cleavage with apoptosis (Pizzoferrato et al, 2004;Kim et al, 2004). We provide evidence of mitochondrial involvement through immunoblotting of bid, Smac/DIABLO and cytochrome c. Use of specific and pan-caspase inhibitors point to the role of caspase-8 as the apical caspase in the IRF-1 apoptotic pathway.…”

“…We previously demonstrated the role of caspases in IRF-1 mediated apoptosis, but did not completely block apoptosis with pan-caspase inhibitor, which could imply a non-caspase mediated or necrotic pathway (Pizzoferrato et al, 2004;Kim et al, 2004). However, in our current study, we were able to completely abrogate apoptosis using the pan-caspase inhibitor ZVAD, which appears to confirm that in our current model not only is the cell death observed occurring through apoptosis, but also that it is completely caspase mediated.…”

Interferon-regulatory factor-1 is critical for tamoxifen-mediated apoptosis in human mammary epithelial cells

“…This finding is not completely unexpected. Recent reports have highlighted the ability of IRF-1 to mediate growth arrest and apoptosis in breast cancer cell lines (Kim et al, 2002(Kim et al, , 2004Hoshiya et al, 2003). In addition, evidence suggests that IRF-1 plays a role in mammary homeostasis, as IRF-1 is critical for mammary gland involution and loss of expression of IRF-1 is an early event in mammary carcinogenesis (Doherty et al, 2001).…”

“…Specifically, IRF-1 promotes apoptosis associated with caspase-1 activation (Tamura et al, 1995;Romeo et al, 2002). More recently, IRF-1 has been shown to be a tumor suppressor and critical for mammary gland involution (Yim et al, 1997;Nozawa et al, 1999;Hoshiya et al, 2003;Kim et al, 2004). Studies have also shown that IRF-1 expression is lowered or the gene is mutated in multiple cancers, including breast cancer (Doherty et al, 2001;Tzoanopoulos et al, 2002).…”

“…Recently, overexpression of IRF-1 in two mouse breast cancer cell lines has also shown to activate caspase-3 (Kim et al, 2004). We have previously shown that caspase-3 is activated by 1.0 mM Tam in acutely damaged HMEC-E6 cells starting at 6 h, maximally at 24 h, and precedes the appearance of marginated chromatin (early effector-phase apoptosis), first observed at 12 h .…”