Tumor suppressor IRF-1 mediates retinoid and interferon anticancer signaling to death ligand TRAIL

Abstract

Retinoids and interferons are signaling molecules with pronounced anticancer activity. We show that in both acute promyelocytic leukemia and breast cancer cells the retinoic acid (RA) and interferon signaling pathways converge on the promoter of the tumoricidal death ligand TRAIL. Promoter mapping, chromatin immunoprecipitation and RNA interference reveal that retinoid-induced interferon regulatory factor-1 (IRF-1), a tumor suppressor, is critically required for TRAIL induction by both RA and IFNgamma. Exposure of breast cancer cells to both antitumor agents results in enhanced TRAIL promoter occupancy by IRF-1 and coactivator recruitment, leading to strong histone acetylation and synergistic induction of TRAIL expression. In coculture experiments, pre-exposure of breast cancer cells to RA and IFNgamma induced a dramatic TRAIL-dependent apoptosis in heterologous cancer cells in a paracrine mode of action, while normal cells were not affected. Our results identify a novel TRAIL-mediated tumor suppressor activity of IRF-1 and suggest a mechanistic basis for the synergistic antitumor activities of certain retinoids and interferons. These data argue for combination therapies that activate the TRAIL pathway to eradicate tumor cells.

Figure 1

Mapping of RA-responsive sites in the TRAIL promoter region. (A) Illustration of pTRL reporter genes containing the TRAIL upstream regulatory region and site-specific mutants derived from pTRL3. (B) DNase I hypersensitive mapping of the TRAIL locus schematically depicted below. Nuclei of vehicle- (‘−') or RA-treated NB4 and SK-BR-3 cells were treated with increasing amounts of DNase I (0, 2 and 4 U; indicated at the top). Hypersensitive regions are shown as DHS I, II and III. The asterisk indicates that DHS I is the only RA-induced site in both NB4 and SK-BR-3. (C) Luciferase activity in SK-BR-3 transiently transfected with the indicated pTRL reporters in the presence (black bars) or absence (white bars) of RA. (D) Transfection experiment as in (C) but using pTRL3 derivatives with mutated response elements. (E) EMSA with the TRAIL IRF-E and nuclear extracts from SK-BR-3 cells treated with RA or IFNγ, as indicated below. Complexes (arrow) were competed with cold probe or supershifted with antibodies (triangles) directed against IRF-1, IRF-2 and STAT1, as depicted at the top.

Figure 2

RA-induced IRF-1 is sufficient to induce TRAIL promoter activity. (A) SK-BR-3 cells were treated with RA for 3 h and ChIPs performed using antibodies directed against acetylated histones H3 (lanes 3 and 4) or H4 (lanes 5 and 6). Immunoprecipitated chromatin was analyzed by PCR with primers specific for promoter-proximal regions of the IRF-1 or GAPDH genes. (B) SK-BR-3 and NB4 cells were treated with IFNα, IFNγ or RA as indicated. Shown are immunoblots generated with anti-IRF-1, anti-IRF-2 and anti-STAT1 antibodies. (C) Semiquantitative RT–PCR performed with primers for TRAIL or GAPDH mRNA using total RNA of MCF7 (lanes 1–4) or SK-BR-3 (lanes 5–8) either treated with RA for 48 h (lanes 2 and 6) or transiently transfected with empty (pCDNA3; lanes 1 and 5) or IRF-1 expression vector (2 μg, lanes 3 and 7; or 5 μg, lanes 4 and 8). (D) SK-BR-3 cells were transiently cotransfected with the indicated pTRL reporters and either pCDNA3 (white bars) or the pCDNA3-based IRF-1 expression vector (black bars).

Figure 3

IRF-1 is recruited to the TRAIL promoter in vivo and required for RA-induced TRAIL expression. (A, B) ChIP assays using antibodies to IRF-1 and IRF-2 (indicated at the top) and SK-BR-3 (A) or NB4 (B) cells treated with RA for 36 h. Immunoprecipitated chromatin was analyzed by PCR using primers specific for the TRAIL, HSP70 or GAPDH promoters. (C) siRNA to IRF-1 specifically knocks down expression of IRF-1 protein. H3396 breast cancer cells were mock transfected or transfected with siRNA to IRF-1 at 200 nM and treated with RA for 36 h. Shown is the corresponding immunoblot using antibody to IRF-1 or actin. (D) H3396 cells were transfected and treated as in (C) and total RNA was isolated. Semiquantitative RT–PCR was performed with primers specific for TRAIL or GAPDH mRNAs.

Figure 4

RA and IFNγ synergize to induce TRAIL. (A) Luciferase activities of SK-BR-3 cells transfected with pTRL reporters and treated with RA and/or INFγ as depicted. (B) H3396 cells were treated with RA, IFNγ or both as indicated and ChIP assays were performed using anti-IRF-1 antibodies. Immunoprecipitated chromatin was analyzed by PCR using primers specific for the TRAIL or GAPDH promoters. A representative ChIP experiment analyzed on an ethidium bromide gel and data from three independent ChIP experiments analyzed by real-time PCR are shown. (C, D) H3396 cells were treated as in (B) and ChIP assays were performed using no antibody (‘No Ab') or anti-CBP or anti-acetylhistone H3 antibodies. The graphs depict real-time PCR data as percent input immunoprecipitated. (E, F) SK-BR-3 cells were treated as in (A) and semiquantitative RT–PCR (E) with primers for TRAIL and GAPDH or Western blot analyses (F) with anti-TRAIL or anti-actin antibodies were performed as indicated.

Figure 5

RA and IFNγ cooperatively recruit IRF-1 and PolII to the TRAIL promoter. (A) Western blot of IRF-1 expression in H3396 cells treated with RA, IFNγ or both as indicated. (B) H3396 cells were treated as indicated and ChIP assays were performed using anti-IRF-1 antibodies. Immunoprecipitated chromatin was analyzed by real-time PCR using primers specific for the TRAIL promoter. Data from two independent ChIP experiments (expressed as % input) are shown. (C) Similar to (B) but anti-PolII antibodies were used to perform the ChIP. (D) H3396 cells were treated as depicted, RNA was isolated and quantitative PCR was performed with cDNA (diluted 1/40) from a reverse transcription reaction using 3 μg RNA (representative of three experiments).

Figure 6

RA and IFNγ synergize to kill leukemic, but not normal, T cells in a paracrine mode of action involving TRAIL. (A) Coculture experiment for paracrine death induction performed as outlined at the top. SK-BR-3 effector cells were cocultured for 72 h with Cell Tracker-labeled Jurkat target cells at a 10:1 ratio in the absence or presence of RA and/or IFNγ. Death induced in target cells was analyzed by PI incorporation and FACS analysis; paracrine killed target cells are gated to the right top quadrants, and the left quadrants gate the Cell Tracker-negative effector cells. Numbers indicate the percentage of PI-positive and -negative target cells. (B) Bar graph showing the percentage of dead Jurkat cells in a representative experiment performed as in (A) with SK-BR-3 effector cells in the absence (gray bars) or presence of RA and INFγ (black bars) and presence of neutralizing receptor–Fc chimeras as shown; similar results were obtained in three independent experiments. Note that only the TRAIL receptor chimera inhibits paracrine death. (C) Leukemia cell-selective paracrine death induction analyzed by coculturing SK-BR-3 effector and either Jurkat leukemia (upper panels) or normal CD4+ T (lower panels) target cells as outlined at the top. The effector cells were treated with RA and INFγ for 48 h before labeled target cells were cocultured for a further 24 h at a two-fold excess of effector cells. FACS data are presented as in (A); only the percentage of paracrine death is given.

Figure 7

Sketch illustrating the convergence of three different anticancer signals on the TRAIL promoter. TRAIL is integrated in a tumor suppressive signaling network that is triggered by RA, IFN and HDAC inhibitors. RA and IFN synergistically induce expression of IRF-1, which activates the promoter of TRAIL through the ISRE and IRF-E sites. HDAC inhibitors lead to acetylation of histone and SP1 family members, thereby inducing TRAIL promoter activation. IFN also induces p53 expression, which stimulates the TRAIL receptor DR5 and the cell cycle inhibitor p21 whose expression is also upregulated by RA and HDAC inhibitors, thus promoting cell cycle arrest. Pink boxes indicate tumor suppressors; CoA, coactivator.