ABSTRACT
Interferon (IFN) regulatory factor-1 (IRF-1) is a transcription factor that has apoptotic anti-tumor activity. In breast cancer cell types, IRF-1 is implicated in mediating apoptosis by both novel and established anti-tumor agents, including the anti-estrogens tamoxifen and faslodex. Here we demonstrate that in MDA468 breast cancer cells, apoptosis by IFN-gamma is mediated by IRF-1 and IFN-gamma, and IRF-1-induced apoptosis is caspase-mediated. IRF-1 induction results in cleavage of caspase-8, -3 and -7, and application of caspase inhibitors attenuate activated cleavage products. IRF-1-induced apoptosis involves caspase-8 since apoptosis is significantly decreased by the caspase-8-specific inhibitor IETD, c-FLIP expression and in caspase-8-deficient cancer cells. Furthermore, we demonstrate that IRF-1-induced apoptosis requires fas-associated death domain (FADD) since dominant-negative FADD expressing cells resist IRF-1-induced apoptosis and activated downstream products. Immunofluorescent studies demonstrate perinuclear colocalization of FADD and caspase-8. Despite the known role of FADD in mediating death-ligand induced apoptosis, neutralizing antibodies against classical death receptors do not inhibit IRF-1 induced apoptosis, and no secreted ligand appears to be involved since MDA468 coincubated with IRF-1 transfected cells do not apoptose. Therefore, we demonstrate that IRF-1 induces a ligand-independent FADD/caspase-8-mediated apoptosis in breast cancer cells.
Subject(s)
Apoptosis/drug effects , Breast Neoplasms/pathology , Fas-Associated Death Domain Protein/metabolism , Interferon Regulatory Factor-1/metabolism , Interferon-gamma/pharmacology , Signal Transduction , fas Receptor/metabolism , Breast Neoplasms/metabolism , Caspase 3/metabolism , Caspase 8/metabolism , Caspase 9/metabolism , Caspases/metabolism , Fas-Associated Death Domain Protein/antagonists & inhibitors , Fas-Associated Death Domain Protein/genetics , Fluorescent Antibody Technique , Genes, Dominant , Humans , Immunoblotting , Ligands , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Subcellular Fractions , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Cells, CulturedABSTRACT
Stimulation of quiescent mouse fibroblasts with TGF-beta1 and certain other growth factors result in cooperative activation of tissue factor (TF) gene transcription, an event accompanied by the rapid entry of c-Fos into specific AP-1 DNA-binding complexes (Felts et al. (1995) Biochemistry 34, 12355-12362). Here, we demonstrate that the ability of TGF-beta1 to synergistically activate TF transcription in serum-stimulated fibroblasts is dependent upon both c-Fos and a promoter-specific factor with functional properties characteristic of transcriptional coactivators. Inhibition of TF promoter activity by an adenovirus E1A mutant deleted in an essential CREB binding protein (CBP) interaction domain suggests that this factor is distinct from the CBP/p300 family of transcriptional coactivators. Importantly, the ability of this factor to mediate molecular interactions with c-Fos required for transcriptional synergism is directly linked to TGF-beta1 signaling. These data suggest a model in which a component of the TF basal transcription complex functions to integrate multiple signaling pathways required for full transcriptional activation of TF in fibroblasts.
Subject(s)
Adenovirus E1A Proteins/physiology , Proto-Oncogene Proteins c-fos/physiology , Trans-Activators , Transforming Growth Factor beta/physiology , Animals , CREB-Binding Protein , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/metabolism , Drug Synergism , Gene Expression Regulation , Mice , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Signal Transduction , Thromboplastin/genetics , Transcription Factors/metabolismABSTRACT
Tissue factor (TF), the cellular initiator of the protease blood coagulation cascade, has been shown to be expressed in a variety of solid tumors, particularly those of epithelial origin. However, the mechanisms that mediate TF expression in tumors, as well as the clinical implications of this expression, remain largely unknown. In this study, we examined the cytological distribution of TF in normal human breast tissue and breast carcinomas. Epithelial cells exhibited TF immunoreactivity with little obvious correlation with malignant progression from in situ lesions to invasive cancer. However, there was a strong correlation between progression to invasive cancer and the expression of TF antigen in cellular components of the stroma. TF-positive cells were particularly abundant in close proximity to infiltrating tumor cells and included both macrophages and myofibroblasts, as determined by double-immunofluorescent staining for TF and cell type-specific marker proteins. Double-immunofluorescent staining for TF and transforming growth factor beta (TGF-beta) revealed TGF-beta immunoreactivity both in tumor cells and in the extracellular matrix surrounding TF-positive stromal cells. To test the role of carcinoma cell-derived growth factors in the regulation of stromal cell TF activity, we examined the ability of conditioned media (CM) from breast carcinoma cell lines to stimulate TF activity in myofibroblast-like cells in vitro. Extracts from myofibroblasts exposed to CM displayed strong TF procoagulant activity. However, extracts from cells exposed to unconditioned media or CM pretreated with anti-TGF-beta antibodies did not. The induction of TF activity was also observed upon treatment of indicator cells with recombinant TGF-beta isoforms. Collectively, these data indicate that the recruitment and/or activation of TF-expressing stromal cells is an early event in progression to invasive breast cancer and likely occurs, in part, as a paracrine response to tumor cell-derived members of the TGF-beta family of growth factors.
