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Cancer Biology & Therapy logoLink to Cancer Biology & Therapy
. 2015 May 26;16(7):1029–1041. doi: 10.1080/15384047.2015.1046646

IRF-1 inhibits NF-κB activity, suppresses TRAF2 and cIAP1 and induces breast cancer cell specific growth inhibition

Michaele J Armstrong 1, Michael T Stang 1, Ye Liu 1, Jin Yan 5, Eva Pizzoferrato 2,3,4,†, John H Yim 5,†,*
PMCID: PMC4622679  PMID: 26011589

Abstract

Interferon Regulatory Factor (IRF)-1, originally identified as a transcription factor of the human interferon (IFN)-β gene, mediates tumor suppression and may inhibit oncogenesis. We have shown that IRF-1 in human breast cancer cells results in the down-regulation of survivin, tumor cell death, and the inhibition of tumor growth in vivo in xenogeneic mouse models. In this current report, we initiate studies comparing the effect of IRF-1 in human nonmalignant breast cell and breast cancer cell lines. While IRF-1 in breast cancer cells results in growth inhibition and cell death, profound growth inhibition and cell death are not observed in nonmalignant human breast cells. We show that TNF-α or IFN-γ induces IRF-1 in breast cancer cells and results in enhanced cell death. Abrogation of IRF-1 diminishes TNF-α and IFN-γ-induced apoptosis. We test the hypothesis that IRF-1 augments TNF-α-induced apoptosis in breast cancer cells. Potential signaling networks elicited by IRF-1 are investigated by evaluating the NF-κB pathway. TNF-α and/or IFN-γ results in decreased presence of NF-κB p65 in the nucleus of breast cancer cells. While TNF-α and/or IFN-γ can induce IRF-1 in nonmalignant breast cells, a marked change in NF-κB p65 is not observed. Moreover, the ectopic expression of IRF-1 in breast cancer cells results in caspase-3, -7, -8 cleavage, inhibits NF-κB activity, and suppresses the expression of molecules involved in the NF-κB pathway. These data show that IRF-1 in human breast cancer cells elicits multiple signaling networks including intrinsic and extrinsic cell death and down-regulates molecules involved in the NF-κB pathway.

Keywords: apoptosis, breast cancer, IRF-1, IAP, NF-κB, p53, tumor suppressor

Abbreviations

Ad

adenovirus

β-gal

β-galactosidase

Cdk

cyclin-dependent kinase

cIAP1

c-inhibitor of apoptosis

DISC

death-inducing signaling complex

DMEM

Dulbecco's Modified Eagle's Medium

EGFP

enhanced green fluorescent protein

FADD

fas-associated death domain

FBS

Fetal Bovine Serum

FITC

fluorescein isothiocyanate

FLICE

fas-associated death domain protein interleukin-1 β-converting enzyme

cFLIP

cellular FLICE inhibitory protein

DR

death receptor

ER

estrogen receptor

IFN-β

interferon-β

IFN-γ

interferon-gamma

IκB

Inhibitory kappaB

IKK

IκB, kinase complex

IRF-1

interferon regulatory factor-1

MOI

multiplicity of infection

MTT

methylthiazoltetrazolium

NEMO

NF-κB essential modulator

NF-κB

nuclear factor of kappa Beta

RIP1

receptor interacting protein 1

SCID

severe combined immunodeficiency

siRNA

small interfering RNA

Smac/DIABLO

Second mitochondria-derived activator of caspase/Direct IAP-binding protein with low pI

STAT

signal transducer and activator of transcription

TNF-α

tumor necrosis factor-α

TNFR

tumor necrosis factor receptor

TRADD

TNF receptor associated protein with a death domain

TRAF2

tumor necrosis factor receptor-associated factor 2

TRAIL

tumor necrosis factor-related apoptosis-inducing ligand

XIAP

X-linked inhibitor of apoptosis protein

Introduction

Interferon Regulatory Factor (IRF)-1 is a transcription factor that was originally identified as a regulator of the human interferon (IFN)-β gene.1 It is involved in both innate and adaptive immunity,2 and IRF-1 is a critical mediator of tumor cell death induced by retinoic acid,3,4 tamoxifen,5 faslodex6 and Mullerian inhibiting substance.7 Moreover, IFN-γ has been shown to have a direct growth inhibitory effect on tumor cells and IFN-γ elicits tumor cell death that can be mediated by IRF-1.8-15

Decreased expression of IRF-1 has been observed by immunohistochemistry studies in ductal carcinoma in situ specimens when compared with adjacent normal breast tissue.16 Diffuse cytoplasmic staining of IRF-1 was observed in cells lining the ducts in normal breast epithelium. In cancerous cells, diffuse staining was observed in both the cytoplasm and nucleus. Invasive breast cancer specimens exhibited IRF-1 expression similar to normal breast tissue.16 Specimens from 6 of 7 invasive breast cancer patients that had lymph node metastasis were IRF-1 negative. The absence of IRF-1 expression in these patients correlated with the presence of lymph node metastasis.16 In other studies, statistical analysis showed a negative correlation between IRF-1 expression and tumor grade in tissue microarrays containing specimens of clinically defined invasive breast carcinoma.17 Tissue microarrays constructed using untreated breast cancer tumors showed primarily IRF-1 staining in the cytoplasm.18 Moreover, IRF-1 mRNA was found to be lower in an antiestrogen unresponsive, ER+ breast cancer tumor cell line when compared with antiestrogen-sensitive, ER+ cell lines. IRF-1 mRNA could be induced by the antiestrogen tested in the ER+ antiestrogen-sensitive breast cancer cells, but IRF-1 could not be induced in the antiestrogen-resistant breast cancer cell line.6 Cancer gene expression microarray datasets were assessed to evaluate IRF-1 mRNA and breast cancer clinical outcomes. Low mRNA expression was strongly correlated with risk of recurrence and risk of death.19

IRF-1 can revert NIH3T3 cells transformed by the oncogenes c-myc and fosB to a non-malignant phenotype showing its tumor suppressive activity.20 IRF-1 inhibits tumor growth6,21-23 and the ectopic expression of IRF-1 results in tumor cell death.24-26 We have shown that the ectopic expression of IRF-1 in human breast cancer cell lines results in tumor cell death associated with the downregulation of survivin.24 We also showed that the combination of IRF-1 and adriamycin on the total number of apoptotic and necrotic cells is additive.24 Moreover, we have shown that the intratumoral treatment of tumor bearing mice with Ad-IRF-1 results in the inhibition of tumor growth in vivo in both xenogeneic and syngeneic mouse model systems of breast carcinoma.22,24 Resected tumor specimens had a predominant IRF-1-positive, survivin-negative phenotype.24

In addition, studies have shown that IRF-1 plays a pivotal role in Fas-mediated apoptosis by IFN-γ in renal cell carcinoma cells.27 IRF-1 induction by IFN-γ mediates the synergistic tumor cell death that is observed in human cervical cancer cells treated with IFN-γ and TNF-α.28 IFN-α, however, induces human bladder cancer cell death by a STAT-1/IRF-1-dependent induction of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).29 Similarly IFN-γ30 or IFN-γ in combination with retinoic acid31 results in IRF-1-mediated induction of TRAIL and subsequent breast cancer cell death. Moreover, the induced TRAIL elicits apoptosis in a paracrine and tumor selective manner in cells cocultured with these breast cancer cells.31 Paracrine apoptosis is inhibited by the addition of neutralizing TRAIL receptor-Fc chimeras.31

We have shown that human breast cancer cells infected with Ad-IRF-1 and subsequently cultured with TRAIL results in apoptotic cell death. By using neutralizing antibodies to Fas, TNFR-1, DR4 and/or DR5, we showed that secretion of TNF, TRAIL, and FasL did not appear to be involved in IRF-1 induced apoptosis.32 Moreover, apoptosis was not observed in transwells indicating that a paracrine effect from soluble factors is not involved in mediating tumor cell death.

Our previous studies showed caspase cleavage in human breast cancer cells that express IRF-1 and cleaved bid, cytochrome c, and Smac/DIABLO were also released into the cytosol.32 Caspase-8 is likely the apical caspase in IRF-1 mediated apoptosis and siRNA against caspase-8 resulted in a statistically significant attenuation of apoptosis.32

Recently, we have shown that the ectopic expression of IRF-1 results in the induction of the cyclin-dependent kinase inhibitor p21 and G1 cell cycle arrest in human cancer cells.33 Reduced expression of the cyclin dependent kinases Cdk2, Cdk4, cyclin E, and the transcription factor E2F1, were also observed in human breast cancer cells.33 Cdc-2 and cyclin B1, known to regulate survivin expression were also decreased in IRF-1 expressing breast cancer cells. While p21 mediates G1 cell cycle arrest, p21 does not play a direct role in the down-regulation of survivin. Our data suggest that IRF-1 may directly regulate survivin expression.33

In this current report, we begin to investigate the effect of IRF-1 in human nonmalignant breast cells. We evaluate growth inhibition and IRF-1-induced cell death in nonmalignant human breast cells and compare these results to breast cancer cells. Despite up to 10-fold increases in the multiplicity of infection (MOI), profound growth inhibition and cell death is not observed in nonmalignant cells when compared with breast cancer cells. Moreover, we show that breast cancer cells treated with TNF-α or IFN-γ induces IRF-1 expression and human breast cancer cells cultured with both IFN-γ and TNF-α enhances cell death compared with cells cultured with either IFN-γ or TNF-α alone. Abrogation of IRF-1 expression by siRNA, decreases the cell death observed with IFN-γ and TNF-α. Moreover, the ectopic expression of IRF-1 results in enhanced breast cancer cell death when these breast cancer cells are cultured with TNF-α.

The vast majority of studies have focused on the interaction of IRF-1 and NF-κB in the inflammatory response.34-39 Few studies, however, have addressed their interaction in apoptosis. It has been shown that transfection of IRF-1 in human ME-180 cervical cancer cells resulted in inhibition of TNFα-induced NF-κB activity.28 In addition, ME-180 cervical cancer cells cultured with all-trans retinoic acid results in apoptotic cell death.40 Moreover, retinoic acid-induced IRF-1 may be mediated in part by NF-κB.41 In this current report, we begin to delineate the precise mechanisms of the induction of human breast cancer cell death by evaluating NF-κB expression and activity. Our data show that NF-κB is expressed in human breast cancer cell lines and the ectopic expression of IRF-1 decreases NF-κB activity in these human breast cancer cells. TNF-α or IFN-γ induces IRF-1 expression in both human breast cancer cell lines and the spontaneously immortalized nonmalignant human breast cells. While NF-κB p65 is decreased in the nuclear fraction of human breast cancer cells cultured with TNF-α, IFN-γ, or the combination of both TNF-α and IFN-γ, a profound change of NF-κB p65 is not observed in nonmalignant breast cells. Caspase-3, -7, and -8 cleavage is observed in the breast cancer cells that express IRF-1. The expression of cIAP1, XIAP, TRAF2, TRADD, and FLIP are also reduced in IRF-1 expressing breast cancer cells. Collectively, these results show that IRF-1 induces multiple signaling networks that promote intrinsic and extrinsic cell death and the downregulation of molecules involved in the NF-κB pathway.

Results

IRF-1 selectively inhibits human breast cancer growth

To evaluate the effects of IRF-1 expression in both human breast cancer cell lines, and nonmalignant human breast cells, we selected the MCF-10F spontaneously immortalized nonmalignant human breast cell line, and the human breast cancer cell lines, ZR75-1, MDA-MB-468, and SK-BR-3. Since IRF-1 expression is low in unstimulated breast cancer cell lines and nonmalignant breast cells, the effect of IRF-1 expression on cell growth and cell death can be effectively evaluated. Cells were infected with the control Ad-Ψ5 or Ad-IRF-1 and cell growth inhibition was measured by MTT assays. The selected MOIs were based on experiments using Ad-EGFP that resulted in >95 % EGFP positive cells. Growth inhibition of at least 40 % was calculated in these breast cancer cell lines when compared to the Ad-Ψ5 infected control cell cohorts (Fig. 1A). Growth inhibition was also evaluated in the MCF-10F, spontaneously immortalized nonmalignant human breast cell line. A change in growth was not observed in the nonmalignant human breast cell line (Fig. 1A). Cells were infected with varying MOIs to determine the appropriate MOI that results in equal expression of IRF-1 protein (Fig. 1B) among the cell lines. Cells were infected with a range of MOIs and cellular lysates were utilized for immunoblotting. The MCF-10F, MDA-MB-468 and SK-BR-3 cell lines were subsequently infected at MOIs of 50, 10, and 25 respectively, and growth inhibition was evaluated. Figure 1C shows that growth inhibition of at least 30 % is observed in both the MDA-MB-468 and SK-BR-3 human breast cancer cell lines, while the nonmalignant MCF-10F breast cell line does not undergo growth inhibition despite comparable IRF-1 protein expression (Fig. 1C). Experiments were also conducted utilizing the HMEpC human nonmalignant mammary epithelial cells and the MDA-MB-468 breast cancer cell lines. The HMEpC and MDA-MB-468 cells infected with MOIs of 50 and 10 respectively resulted in comparable IRF-1 protein expression (Fig. 1D). Similar to the MCF-10F spontaneously immortalized nonmalignant human breast cell line, profound growth inhibition was not observed in the IRF-1 infected HMEpC mammary epithelial cell cohort at the 48 h time point (Fig. 1E). A growth inhibition of 4.16 % is observed in the Ad-Ψ5 infected control cell cohort when compared with the uninfected HMEpC control cells. A growth inhibition of 1.66% was computed for Ad-IRF-1 expressing HMEpC cells compared with the uninfected HMEpC control cells. In contrast, tumor cell growth inhibition of 3.11 % and 24.38 % is observed in the Ad-Ψ5 control and Ad-IRF-1 infected MDA-MB-468 human breast cancer cells respectively when compared with the uninfected control MDA-MB-468 cancer cell cohort (Fig. 1E).

Figure 1.

Figure 1.

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IRF-1 selectively inhibits human breast cancer growth. (A) The spontaneously immortalized nonmalignant human breast cell line, MCF-10F, and the human breast cancer cell lines ZR75-1, MDA-MB-468, and the SK-BR-3 were infected with the control Ad-Ψ5 or Ad-IRF-1, at multiplicities of infection (MOIs) 25, 100, 10, and 10 respectively. Cell growth inhibition was measured by MTT assays as described in Material and Methods. (B) Cells were infected at varying MOIs of Ad-IRF-1 and cells were harvested 24 h later. Cellular lysates were prepared for immunoblotting as described in Material and Methods. IRF-1 expression was evaluated by immunoblotting in the MCF-10F, MDA-MB-468, and the SK-BR-3 cell lines. (C) The MCF-10F, MDA-MB-468, and SK-BR-3 cell lines were subsequently infected at MOIs of 50, 10, and 25 respectively. MTT assays were conducted and cell growth inhibition was calculated as described in Material and Methods. (D) HMEpC and MDA-MB-468 cells were infected at varying MOIs and cells were harvested for immunoblotting as described in Material and Methods. (E) HMEpC and MDA-MB-468 cells were infected at MOIs of 50 and 10 respectively. Cell growth inhibition assays were conducted as described in Material and Methods. MTT was added 48 h post infection.

IRF-1 selectively induces apoptosis in human breast cancer

Previously, we have shown that the ectopic expression of IRF-1 results in apoptotic cell death in human breast cancer cell lines.24 In this current report, cell death was evaluated in the MDA-MB-468 human breast cancer cell line, the nonmalignant human mammary epithelial cells HMEpC, and the spontaneously immortalized nonmalignant human breast cell line MCF-10F. MOIs of 100 were used to infect the MCF-10F and HMEpC cell lines to increase IRF-1 protein expression in these breast cells compared with the MDA-MB-468 and SK-BR-3 breast cancer cell lines (Fig. 2A and B) that were infected with much lower MOIs. There is more than a 4-fold increase in the number of cells in the annexin V apoptotic quadrant and a more than a 3-fold increase in the annexin V/propidium iodide-positive cells at the 36 h time point in the MDA-MB-468 breast cancer cells that express IRF-1 when compared with the vector control infected cell cohort (Fig. 2A). Cell death is reduced with the addition of the pan-caspase inhibitor ZVAD. There is an approximate 4-fold increase in the number of annexin V positive cells with the ectopic expression of IRF-1 in the spontaneously immortalized nonmalignant human breast cell line, MCF-10F, and a similar increase is not observed in the HMEpC nonmalignant Human Mammary Epithelial Cells (Fig. 2A). Pronounced increases in the number of cells in the PI quadrant were not observed in the nonmalignant cells (Fig. 2A). In contrast to the MDA-MB-468 breast cancer cell line infected with Ad-IRF-1, the number of annexin V/propidium iodide-positive cells in the MCF-10F/Ad-IRF-1 infected cell cohort is comparable to the MCF-10F/Ad-Ψ5 infected and uninfected control cells. Similarly, the number of annexin V/propidium iodide-positive cells in the HMEpC/Ad-IRF-1 infected cell cohort is comparable to the HMEpC/Ad-Ψ5 and uninfected cell cohorts (Fig. 2A). It should be noted that the MDA-MB-468/Ad-IRF-1 cells were harvested at the earlier 36 h time point post infection in order to observe cells in each quadrant, while the nonmalignant cells were harvested at the 48 h time point. While Figure 2A is a representative experiment of multiple experiments, Figure 2B is a comprehensive analysis of all experiments. Cell death experiments were conducted and the results show that despite the increased expression of IRF-1, a significant increase in cell death is not observed in the MCF-10F nonmalignant human breast cell line and the HMEpC nonmalignant human mammary epithelial cells (Fig. 2B). A significant increase in cell death, however, is observed in both the MDA-MB-468 and SK-BR-3 human breast cancer cell lines.

Figure 2.

Figure 2.

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IRF-1 selectively induces apoptotic cell death in human breast cancer cell lines. (A) The nonmalignant human mammary epithelial cells, HMEpC, the spontaneously immortalized nonmalignant human breast cell line MCF-10F, and the human breast cancer cell line MDA-MB-468 were either not infected or infected with the Ad-Ψ5 vector control or Ad-IRF-1 at MOIs of 100,100, 10 respectively. HMEpC and MCF-10F cells were harvested at 48 h post infection while the MDA-MB-468 cells were harvested 36 h post infection to ensure that cells would be observed in each quadrant. Apoptosis was measured by staining with FITC-conjugated annexin V and propidium iodide as described in Materials and Methods. The pan-caspase inhibitor ZVAD was added to the Ad-IRF-1 infected MDA-MB-468 breast cancer cell cohort and apoptosis was evaluated by flow cytometry as described in Materials and Methods. This result is a representative experiment of multiple experiments with comparable results. (B) The HMEpC and MCF-10F cell lines were either not infected or infected with Ad-Ψ5 or Ad-IRF-1 at a MOI of 100. The MDA-MB-468 and SK-BR-3 breast cancer cells were either not infected or infected at a MOI of 5 and 10 respectively. Infected cells were harvested and immunoblotting was performed as described in Materials and Methods. Cells were harvested and cell death assays were conducted as described in Materials and Methods. These experiments were performed in triplicate and comprehensive analyses of all data were conducted. Percent apoptosis indicated in the bar graphs represents the mean percent of gated cells in the early and late apoptotic quadrants. Standard deviation is indicated by error bars. * p<0.0001 by ANOVA for both the MDA-MB-468 and SK-BR-3 breast cancer cell lines infected with Ad-IRF-1 versus uninfected or Ad-Ψ5 infected control cell cohorts.

TNF-α or IFN-γ sensitizes human breast cancer cells to IRF-1 induced cell death

We have shown that IFN-γ results in the induction of IRF-1 and apoptosis in human breast cancer cells.32,33 siRNA against IRF-1 resulted in the reduction of IRF-1 expression and a statistically significant inhibition of IFN-γ induced apoptosis.32 IRF-1 induction by IFN-γ mediates the synergistic tumor cell death that is observed in human cervical cancer cells treated with IFN-γ and TNF-α.28 These results suggest that IRF-1 may also enhance TNF-α induced cell death. To test this hypothesis, cell death was measured in the human breast cancer cell lines, MDA-MB-468 and SK-BR-3, cultured with IFN-γ, TNF-α, or a combination of IFN-γ and TNF-α. Statistically significant increases in cell death are observed in both the MDA-MB-468 and SK-BR-3 breast cancer cells cultured with both IFN-γ and TNF-α when compared with IFN-γ or TNF-α alone (Fig. 3A). Statistically significant increases in cell death were observed in the MDA-MB-468 breast cancer cells cultured with either IFN-γ or TNF-α alone when compared with untreated cells. In contrast, statistically significant increases in cell death were observed in the SK-BR-3 breast cancer cells cultured with TNF-α alone when compared with the untreated cell cohort. A statistically significant increase was not observed in SK-BR-3 cells cultured with IFN-γ alone when compared with the untreated cells (Fig. 3A). Increased apoptosis is observed in these breast cancer cell lines infected with Ad-IRF-1. Statistically significant increases in cell death are computed in Ad-IRF-1 infected breast cancer cells when compared with the uninfected or Ad-Ψ5 infected control cell cohorts (Fig. 3B). Moreover, a statistically significant increase in cell death is observed in MDA-MB-468/Ad-IRF-1 and SK-BR-3/Ad-IRF-1 breast cancer cells cultured with TNF-α compared with the uninfected and Ad-Ψ5 cell cohorts cultured with TNF-α alone (Fig. 3B). It should be noted that a statistically significant increase in cell death is also observed in the Ad-IRF-1/TNF-α cohorts when compared with the Ad-IRF-1 infected breast cancer cell cohorts (Fig. 3B). We also evaluated IRF-1 expression in the human breast cancer cell lines cultured with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ. While IRF-1 expression is not detected by immunoblotting in the MDA-MB-468 breast cancer cells, TNF-α or IFN-γ can induce the expression of IRF-1 in these breast cancer cells (Fig. 3C). Densitometric analyses show that the combination of TNF-α and IFN-γ results in increased protein expression of IRF-1 (Fig. 3C). IRF-1 expression, however, is induced in SK-BR-3 breast cancer cells when cultured with TNF-α (Fig. 3C). Since the combination of IFN-γ and TNF-α resulted in augmented IRF-1 expression and enhanced cell death in the MDA-MB-468 human breast cancer cells, these cancer cells were transfected with siRNA to IRF-1 prior to culture with IFN-γ and TNF-α (Fig. 4A). The results show a statistically significant decrease in cell death in human breast cancer cells transfected with siRNA to IRF-1 and cultured with IFN-γ and TNF-α (Fig. 4B).

Figure 3.

Figure 3.

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TNF-α and/or IFN-γ induces IRF-1 and enhances human breast cancer cell apoptosis. (A) The MDA-MB-468 and SK-BR-3 breast cancer cells were either cultured in medium alone or cultured with IFN-γ, TNF-α, or IFN-γ and TNF-α as described in Materials and Methods. Apoptosis was evaluated as described in Materials and Methods. (B) The MDA-MB-468 or SK-BR-3 breast cancer cells were either not infected or infected with the control Ad-Ψ5 or Ad-IRF-1. Infected cells were subsequently cultured with TNF-α and apoptosis was measured by FITC-conjugated annexin V and propidium iodide as described in Materials and Methods. Percent apoptosis indicated in the bar graphs represents the mean percent of gated cells in the early and late apoptotic quadrants. Standard deviation is indicated by error bars. * = p < 0.05 by unpaired t test. p < 0.01 for the MDA-MB-468/Ad-IRF-1 + TNF-α cohort compared with the MDA-MB-468/Ad-IRF-1 cohort and p < 0.0001 for the SK-BR-3/Ad-IRF-1 + TNF-α compared with SK-BR-3/Ad-IRF-1 breast cancer cohort. (C) The MDA-MB-468 and SK-BR-3 human breast cancer cells were either cultured in media, or cultured with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ as described in Materials and Methods. Cells were harvested after 24 h in culture and immunoblotting was performed as described in Materials and Methods.

Figure 4.

Figure 4.

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Abrogation of IRF-1 results in diminished IFN-γ and TNF-α induced apoptosis. (A) MDA-MB-468 breast cancer cells were either not transfected or transfected with a control siNeg, or siRNA to IRF-1. Untransfected cells were treated with the transfection reagent alone (T.R.) as an additional control. Cells were harvested and immunoblotting was performed as described in Materials and Methods. (B) Abrogation of IRF-1 results in diminished IFN-γ and TNF-α induced apoptosis. MDA-MB-468 human breast cancer cells were transfected with a control siNeg or siRNA to IRF-1. 24 h post transfection the cells were cultured with IFN-γ and TNF-α and cell death was evaluated as described in Materials and Methods. Standard deviation is indicated by error bars. *= p < 0.05 by unpaired t test.

IRF-1 suppresses NF-κB activity

As an anti-apoptotic transcription factor, the upregulation of NF-κB may function as a survival strategy for cancer cells and the activation of NF-κB signaling has been reported in breast cancer cell lines and tumors.42-43 NF-κB is composed of homo and heterodimers of Rel-domain containing proteins that include p65/RelA, RelB, c-Rel, p50/p105 (NF-κB1), p52/p100 (NFκB2). Inactive NF-κB is sequestered in the cytoplasm through its physical association with the inhibitory IκB proteins. The predominant heterodimer is the p50/p65 complex.44,45 NF-κB induction occurs through the phosphorylation and subsequent ubiquitin-dependent degradation of IκB proteins. The dissociation of IκB proteins from NF-κB results in the unmasking of the nuclear localization sequence of p65 which subsequently facilitates the translocation of NF-κB to the nucleus.46 The p65 subunit of NF-κB mediates the transactivation of downstream genes involved in immunoregulation, growth regulation, inflammation, carcinogenesis, and apoptosis.47

NF-κB expression was evaluated in the SK-BR-3 and MDA-MB-468 breast cancer cell lines and the nonmalignant MCF-10F, either uninfected or infected with the Ad-Ψ5 control or Ad-IRF-1 (Fig. 5A). NF-κB p65 is expressed in the MDA-MB-468 and SK-BR-3 human breast cancer cell lines and p65 expression is comparable upon ectopic expression of IRF-1. Moreover, NF-κB is expressed in the MCF-10F cell line (Fig. 5A). Using a NF-κB luciferase reporter, NF-κB activity was evaluated in the human breast cancer cell lines. High levels of NF-κB activity were confirmed in the human breast cancer cell lines (Fig. 5B). NF-κB activity, however, was subsequently decreased in the MDA-MB-468 and SK-BR-3 breast cancer cells infected with Ad-IRF-1 or the Ad-IκB super-repressor that encodes a mutant protein that sequesters NF-κB proteins (Fig. 5B). The expression of proteins involved in the NF-κB pathway was also evaluated. Immunoblotting data show that the protein expression of cIAP1, TRADD, and TRAF2 is decreased in the human breast cancer cells infected with Ad-IRF-1 (Fig. 5C).

Figure 5.

Figure 5.

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Ectopic expression of IRF-1 reduces NF-κB expression and activity in human breast cancer cells. (A) The nonmalignant human mammary epithelial cells HMEpC, the spontaneously immortalized nonmalignant human breast cell line MCF-10F, and the human breast cancer cell lines MDA-MB-468 and the SK-BR-3 were either uninfected or infected with the control Ad-Ψ5 or Ad-IRF-1 as previously described. Cellular lysates were prepared and immunoblotting for NF-κB p65 was performed as described in Materials and Methods. (B) The MDA-MB-468 and SK-BR-3 human breast cancer cells were co-transfected with the NF-κB responsive luciferase reporter construct pELAM-luc and β-gal plasmid pIEPlacZ. 24 h post transfection cells were either not infected or infected with the control Ad-Ψ5, Ad-IRF-1, or Ad-IκB super-repressor in duplicate. 24 h post infection luciferase activity was measured as described in Materials and Methods. (C) Uninfected cells or cells infected with the Ad-Ψ5 control or Ad-IRF-1 at the indicated MOIs were harvested 24 h post infection. Cellular lysates were used for immunoblotting as described in Materials and Methods.

To better understand the role of IRF-1 in the NF-κB signaling pathway in human breast cancer cell lines and nonmalignant breast cells, the human breast cancer cell line MDA-MB-468 and the spontaneously immortalized nonmalignant human breast cell line MCF-10F were cultured with TNF-α, IFN-γ, or the combination of both TNF-α and IFN-γ. Nuclear and cytoplasmic fractions were subsequently isolated and probed for NF-κB p65. By densitometric analyses, a 15% decrease in p65 expression was computed in the nuclear fraction when the human MDA-MB-468 breast cancer cells were cultured with TNF-α (Fig. 6A). Breast cancer cells cultured with IFN-γ, however, resulted in a 34 % decrease in p65 protein expression in the nucleus. A 22% decrease in p65 expression in the nucleus was calculated when the MDA-MB-468 breast cancer cells were cultured with both TNF-α and IFN-γ (Fig. 6A). Similar to the MDA-MB-468 human breast cancer cell line, TNF-α and/or IFN-γ induces IRF-1 expression in the nonmalignant MCF-10F breast cell line (Figs. 3C and 6B). When evaluating NF-κB p65 expression in the nuclear and cytosolic fraction, however, a profound change in expression is not observed in the spontaneously immortalized nonmalignant MCF-10F human breast cells either not treated or cultured with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ (Fig. 6C).

Figure 6.

Figure 6.

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p65 protein expression is decreased in the nuclear fraction of the human breast cancer cell line MDA-MB-468. (A) The human breast cancer cells, MDA-MB-468, were either not treated or cultured with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ. 24 h post treatment, cells were harvested and nuclear and cytosolic fractions were isolated. (B) The spontaneously immortalized nonmalignant human breast cell line, MCF-10F, was either not treated or cultured with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ as described in Materials and Methods. 24 h post culture, cells were harvested and immunoblotting was performed as described in Materials and Methods. (C) The MCF-10F cells were either not treated or cultured with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ. 24 h post treatment, cells were harvested and nuclear and cytosolic fractions were isolated. Immunoblotting for NFκB p65 in nuclear and cytosolic fractions was performed as described in Materials and Methods.

FLIP, TRAF2, and XIAP expression is suppressed by ectopic IRF-1 expression

To investigate molecules involved in the NF-κB pathways and molecules involved in both the intrinsic and extrinsic apoptotic pathways, both the SK-BR-3 and MDA-MB-468 human breast cancer cell lines were either uninfected or infected with Ad-Ψ5 or Ad-IRF-1. Uninfected or infected cells were also cultured with TNF-α. Caspase-8 cleavage was confirmed in both the MDA-MB-468 and SK-BR-3 human breast cancer cell lines infected with Ad-IRF-1 (Fig. 7A). In addition to the reduced expression of TRAF2 and TRADD (Fig. 5C), suppression of FLIP is also observed in the SK-BR-3/Ad-IRF-1 with or without TNF-α (Fig. 7A). The reduction of FLIP is more pronounced in the MDA-MB-468/Ad-IRF-1 cells cultured with TNF-α (Fig. 7A). Caspase-3 and caspase-7 cleavage was also evaluated in the MDA-MB-468 human breast cancer cells. Caspase-3 and caspase-7 cleavage was observed in the Ad-IRF-1 infected cells or Ad-IRF-1 infected cells cultured with TNF-α (Fig. 7B). In addition, the expression of the X-linked inhibitor of apoptosis, XIAP, expression was evaluated in human breast cancer cells expressing IRF-1 (Fig. 7C). While XIAP is expressed in MDA-MB-468 uninfected or Ad-Ψ5 infected cells, XIAP protein expression is decreased in the MDA-MB-468/Ad-IRF-1 infected cell cohort. The most pronounced decrease in XIAP expression is observed at the 48 h time point post infection (Fig. 7C).

Figure 7.

Figure 7.

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FLIP protein expression is suppressed by Ad-IRF-1 infection in human breast cancer cells. (A) SK-BR-3 and MDA-MB-468 human breast cancer cells were either uninfected or infected with Ad-Ψ5 or Ad-IRF-1 at a MOI of 50 and 25 respectively and cultured with or without 500 U/mL of TNF-α. After 24 h, cells were harvested and cellular lysates were loaded and utilized for immunoblotting as described in Materials and Methods. Multiple experiments were run on the same gel and at the same time, and the relevant results are presented. (B) MDA-MB-468 human breast cancer cells were either uninfected or infected with Ad-Ψ5 or Ad-IRF-1 at a MOI of 25 and cultured with or without 500 U/mL of TNF-α. After 24 h, cells were harvested and cellular lysates were loaded and utilized for immunoblotting as described in Materials and Methods. (C) XIAP expression is decreased in Ad-IRF-1 infected human breast cancer cells. MDA-MB-468 human breast cancer cells were either uninfected or infected with Ad-Ψ5 or Ad-IRF-1 and immunoblotting was conducted as described in Materials and Methods.

Discussion

We have shown that the ectopic expression of IRF-1 in human breast cancer cells results in tumor cell death and inhibition of tumor growth in vivo in xenogeneic mouse models of breast carcinoma.24 In this current report, we begin to investigate the effect of IRF-1 on normal human mammary epithelial cells and nonmalignant human breast cells. It has been shown that IRF-1 protein is less frequently expressed in tumor tissue from high grade ductal carcinoma in situ or node-positive invasive ductal cancer compared with normal breast epithelium.16 Other studies have detected IRF-1 expression in breast cancer specimens, but expression is predominantly cytosolic, hence, IRF-1 may be potentially inactive.25 In addition, it was shown using IRF-1 knockout mice that IRF-1 may play a role in the suppression of premature epithelial apoptosis during mammary gland involution.48 It is expected, then, that “normal” nonmalignant mammary cells would be more tolerant to the effects of IRF-1 than cancerous cells.

We investigated the effect of ectopic IRF-1 expression in the nonmalignant human mammary epithelial cells, HMEpCs, the spontaneously immortalized nonmalignant human breast cell line, MCF-10F, and human breast cancer cell lines. While the ectopic expression of IRF-1 effectively inhibited tumor growth in the breast cancer cell lines, minimal inhibition of cell growth was observed in the MCF-10F nonmalignant human breast cell line. We repeated these studies by testing MOIs that resulted in equal IRF-1 protein expression. Inhibition of cell growth was not observed in the MCF-10F cells or the HMEpC human mammary epithelial cells despite the increase in MOI. We then evaluated cell death using both the HMEpC nonmalignant human mammary epithelial cells and the MCF-10F spontaneously immortalized nonmalignant human breast cell line. Apoptosis was observed in the breast cancer cell lines, but cell death was not observed in the HMEpC cells. Minimal apoptosis was detected in the MCF-10F cells. Despite augmented MOIs and increased IRF-1 expression in both the MCF-10F and HMEpC cells, increased cell death was not detected in these cells.

IRF-1 is the major mediator of IFN-γ, hence, IRF-1 may also enhance TNF-α induced apoptosis. Statistically significant increases in apoptosis were observed in the MDA-MB-468 breast cancer cells cultured with either IFN-γ or TNF-α when compared to untreated cells, while statistically significant increases in apoptosis were observed in the SK-BR-3 breast cancer cells cultured with TNF-α alone. We also evaluated the expression of human IRF-1 protein in the human breast cancer cells treated with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ. Human IRF-1 was expressed in the MDA-MB-468 breast cancer cells treated with TNF-α or IFN-γ. By immunoblotting, TNF-α alone induced IRF-1 expression in the SK-BR-3 breast cancer cells.

Indeed, our studies showed that apoptosis was increased in human breast cancer cell lines cultured with both IFN-γ and TNF-α, however, the apoptotic response is greater in cells that express IRF-1. A statistically significant increase in apoptosis is also observed in MDA-MB-468/Ad-IRF-1 and SK-BR-3/Ad-IRF-1 breast cancer cells cultured with TNF-α when compared with the breast cancer cells cultured with TNF-α alone or the Ad-IRF-1 infected cell cohorts. When IRF-1 expression was abrogated by siRNA, apoptosis in human breast cancer cells cultured with IFN-γ and TNF-α was diminished. These data further support the contention that IRF-1 promotes TNF-α induced tumor cell death. Similarly, transient transfection of IRF-1 induced cell death of ME-180 cervical cancer cells treated with TNF-α.28

Collectively, the ectopic expression of IRF-1 inhibited breast cancer cell growth and induced tumor cell death that was not observed in nonmalignant human breast cell lines. Moreover, TNF-α or IFN-γ can induce IRF-1 expression in breast cancer cell lines and tumor cell death is observed. To understand the signaling mechanisms that may be elicited by IRF-1, NF-κB expression and activity were evaluated in both human breast cancer cell lines and nonmalignant breast cells. NF-κB activity is associated with tumor cell growth and survival.49 Activation of NF-κB is observed in various tumors including haematopoietic,50,51 breast,52-54 ovarian,52,55 colon,52 pancreatic,56 thyroid,57 bladder,58 and prostate cancers.59,60 High levels of nuclear NF-κ DNA-binding activity comprised of RelA, c-Rel, and p50 are observed in breast cancer cell lines, most primary human breast cancer specimens, and carcinogen-induced mammary tumors in rats.53,54 The activation of NF-κB appears to be an early event in the transformation of human mammary epithelial cells, and NF-κB activation correlates with hormone-independent growth.53

NF-κB p65 expression was observed in both the MDA-MB-468 and SK-BR-3 breast cancer cell lines while low levels were detected in the nonmalignant human breast cell lines. The low level of NF-κB p65 in nonmalignant breast cells, suggest a mechanism by which IRF-1 may selectively elicit tumor cell death. High NF-κB activity was measured in both human breast cancer cell lines using a NF-κB luciferase reporter. IRF-1 expression resulted in decreased NF-κB activity in both the MDA-MB-468 and SK-BR-3 breast cancer cells. NF-κB activity was also decreased in cells infected with the Ad-IκB super-repressor that encodes a mutant protein that sequesters NF-κB proteins. In ME-180 human cervical cancer cells, it was shown that cells cultured with TNF-α resulted in cell survival.28 Treatment of ME-180 cancer cells with a proteasome inhibitor that inhibits NF-κB activation, however, sensitizes these cells to TNF-α-induced apoptosis.28

By densitometric analyses, the MDA-MB-468 breast cancer cells cultured with IFN-γ resulted in IRF-1 expression, tumor cell death, and diminished NF-κB p65 protein translocation to the nucleus. While IRF-1 expression was increased in the TNF-α and the combination of IFN-γ and TNF-α cell cohorts when compared with the MDA-MB-468 breast cancer cells cultured with IFN-γ alone, the expression of NF-κB p65 in the nucleus was less in this cell cohort when compared with TNF-α treated or breast cancer cells cultured with both IFN-γ and TNF-α. These data suggest that while TNF-α or IFN-γ can induce IRF-1 and results in tumor cell death, the signaling pathways that are elicited by TNF-α or IFN-γ may differ.

Similar to the MDA-MB-468 human breast cancer cell line, the antiestrogen-resistant, MCF7/LCC9, human breast cancer cells treated with IFN-γ induces IRF-1 and significantly reduces the transcriptional activity of NF-κβ p65.61 The transcriptional activity of NF-κB p65 is further reduced when breast cancer cells are treated with both IFN-γ and the steroidal antiestrogen fulvestrant ICI.61

IRF-1 was also expressed in the spontaneously immortalized nonmalignant human breast cell line, MCF-10F, cultured with TNF-α, IFN-γ, and the combination of TNF-α and IFN-γ. In contrast to the human breast cancer cells, however, a change in the expression of NF-κB p65 in the nuclear fraction was not observed in this nonmalignant human breast cell line. These data suggest that the induction of IRF-1 does not play a significant role in NF-kB p65 expression in these nonmalignant human breast cells.

NF-κB pathways are composed of both the classical NF-κB pathway initiated by the activation of tumor necrosis factor (TNF) receptor associated factor (TRAF) adapter proteins and subsequent stimulation of the IκB kinase (IKK) complex that contains the IKK1 and IKK2 kinases and the NF-κB essential modulator (NEMO).62-65 Upon phosphorylation and proteosomal degradation of IκB proteins, the NF-κB dimers are released in the cytoplasm and translocate to the nucleus. The alternative pathway requires TRAF degradation and IKK1 activation that phosphorylates the p100-inhibited NF-κB complexes into the p52-containing NF-κB dimers that can now translocate to the nucleus.62-65

Signaling complexes determine whether NF-κB is activated or whether cell death occurs. Signaling through the TNF receptor (TNFR1) enables recruitment of the TNF receptor-associated protein with a death domain (TRADD) and the serine-threonine kinase receptor-interacting protein 1 (RIP1). TRADD subsequently recruits the TRAF2 adaptor protein via its N-terminal TRAF-binding domain.65 TRAF2 can form complexes with the cellular inhibitor of apoptosis proteins cIAP1 and cIAP2. This complex elicits a NF-κB response but does not result in apoptosis.66 The TNFR1-TRADD-TRAF2 complex can also induce NF-κB signaling65 that transcriptionally upregulates anti-apoptotic proteins including cellular inhibitor of apoptosis protein cIAP1 and cIAP2, and cFLIPL (cellular FLICE inhibitory protein).66 A second complex is composed of TRADD that can also interact with Fas-associated death domain protein (FADD) to form the death-inducing signaling complex (DISC).67 FADD mediates the recruitment and activation of procaspase-8 and cleaved caspase-8 initiates a molecular cascade that terminates in apoptosis.66 After recruitment of FADD and caspase-8, whether cell death is elicited is determined based on the expression of the anti-apoptotic proteins cIAP1 and FLIP.66 Caspase-8 activity is inhibited if NF-κB activation promotes the expression of FLIP66 that interferes with caspase-8 activity.

In previous studies, we have shown binding of FADD to caspase-8 and perinuclear colocalization in breast cancer cells expressing IRF-1 by immunofluorescent and coimmunoprecipitation studies.32 In this present report, we evaluated NF-κB activity in the human breast cancer cells, MDA-MB-468 and SK-BR-3, and the protein expression of molecules involved in the NF-κB signaling pathways was assessed. We show that TRADD, TRAF2, FLIP, and cIAP1 are all down-regulated in IRF-1-expressing cells, hence, the expression of molecules involved in the NF-κB signaling cascade is reduced and extrinsic apoptotic cell death pathways appear to be triggered. XIAP can bind to caspase 3, 7, and 9 subsequently abrogating cell death.68 XIAP is also a physiologic activator of NF-κB.69 Since NF-κB promotes the transcriptional upregulation of the same IAPs, an amplification loop promoting cell survival is generated. Our current data show that XIAP expression is reduced in IRF-1 expressing human breast cancer cells. We have shown that survivin is also downregulated in Ad-IRF-1-infected breast cancer cells.24 Since both survivin and XIAP are reduced, the amplification loop is dampened and tumor cell death is enhanced. Collectively, these data suggest that molecules involved in the intrinsic cell death apoptotic pathway are also being triggered to elicit tumor cell death.

Our current results show that while NF-κB is expressed in the human breast cancer cell lines, NF-κB activity is decreased in IRF-1-expressing cells. TRADD, TRAF2, FLIP, cIAP1, and XIAP are down-regulated in IRF-1-expressing breast cancer cells. Caspase-3,-7, and -8 cleavage is observed. IRF-1 induces tumor cell death through the downregulation of the anti-apoptotic proteins cIAP1, survivin, XIAP, FLIP, and subsequent triggering of intrinsic and extrinsic apoptotic pathways. IRF-1 results in the reduced expression of molecules involved in the NF-κB signaling pathway. Taken together, these data support the contention that the introduction of IRF-1 results in breast cancer specific cell death by eliciting multiple signaling networks. The precise delineation and convergence of these signaling networks warrant further investigation.

Materials and Methods

Cell lines and culture

The MDA-MB-468, SK-BR-3, and the ZR75-1 human breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA). The MDA-MB-468 tumor cells were propagated in Dulbecco's Modified Eagle's Medium (DMEM, BioWhittaker Inc., Walkersville, MD) and Ham's F-12 (Invitrogen Life Technologies, Carlsbad, CA) media at a 1:1 ratio supplemented with 10 % fetal bovine serum (FBS), L-glutamine and antibiotics. SK-BR-3 and ZR75-1 breast cancer cells were cultured in RPMI 1640 (BioWhittaker Inc.) with 10% FBS, L-glutamine and antibiotics. The nonmalignant Human Mammary Epithelial Cells, HMEpCs, were purchased from Cell Applications (San Diego, CA) and propagated in complete HMEpC media (Cell Applications Inc.). The spontaneously immortalized nonmalignant human breast cell line, MCF-10F, was propagated in DMEM (BioWhittaker Inc.) with 10% FBS, L-glutamine and antibiotics.

Ad-IRF-1 infection

The MDA-MB-468 and SK-BR-3 human breast cancer cell lines, the non-transformed human MCF-10F nonmalignant human breast cell line, and Human Mammary Epithelial Cells (HMEpC) were either not infected (NI) or infected with the control recombinant adenovirus Ad-Ψ5, or infected with Ad-IRF-1 as has been previously described.24 Cells were infected with multiplicities of infection (MOIs) that result in >95 % expression by Ad-EGFP infection20 and by determining the appropriate MOI that results in equal protein expression of IRF-1.

Apoptosis assay

Apoptosis was measured as has been previously described.24 Briefly, cells were either not infected or infected with the Ad-Ψ5 vector control or Ad-IRF-1 and cultured with or without the pan-caspase inhibitor Z-VAD (100 μM) (Promega, Madison, WI). Cells were harvested at various time points and then stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide using the annexin V-FITC apoptosis detection kit (BD PharMingen, San Diego, CA). Cells were analyzed by flow cytometry using a Beckman Coulter cytometer (Fullerton, CA). Cells were also infected and 4 h post infection, fresh medium containing 500 U/mL of TNF-α (R&D Systems, Minneapolis, MN) was added. Cells were cultured with 1000 U of IFN-γ and apoptosis was evaluated.

Cell growth assay

Cell growth assays were conducted as has been previously described.22 Briefly, cells were infected at the indicated MOIs. Methylthiazoltetrazolium (Sigma-Aldrich Corp., St. Louis, MO) (MTT; 0.5 mg/mL) was added to duplicate wells at 48–72 h time points post infection. Supernatants were subsequently removed and cells were dried. Absorbance was measured by UV spectrophotometry at 550 nm. Cell viability was calculated by the relative dye intensity of the mean of duplicate samples. Percent growth inhibition of Ad-IRF-1 infected cells was calculated according to the formula % = 1− [mean abs (Ad-IRF-1)/mean abs (uninfected cells)] × 100. Percent growth inhibition of the Ad-Ψ5 vector control cell cohorts was calculated according to the formula % = 1−[mean abs (Ad-Ψ5)/mean abs (uninfected cells)] × 100.

Immunoblotting analyses

Immunoblotting was performed as previously described.24 Briefly, the MCF-10F, MDA-MB-468, and SK-BR-3 were infected with Ad-IRF-1 at the indicated MOIs and cells were harvested 24 h post infection. Whole-cell cellular lysates were quantified, separated by SDS-PAGE and transferred onto a nitrocellulose membrane and probed for IRF-1, NF-κB, cIAP1, TRAF2, TRADD, c-FLIPL (Santa Cruz Biotechnology Inc. Santa Cruz, CA), GAPDH, caspase-3, caspase-7, and caspase-8 were purchased from Cell Signaling Technology Inc. (Danvers, MA). Anti-Lamin was purchased from Abcam (Cambridge, MA). The irreversible pan-caspase inhibitor, ZVAD, was purchased by Promega (Madison, WI). Equal loading of proteins was assessed by probing membranes with β-actin (Abcam Inc., Cambridge, MA). Protein bands were visualized using Supersignal (West Pico Chemiluminescent Substrate; Pierce Biotechnology Inc., Rockford, IL) according to the manufacturer's instructions. The bands were subsequently exposed on Kodak film (Eastman Kodak, Rochester, NY) to detect chemiluminescence signals.

Transient transfection of siRNA

Transient transfection of siRNA for human IRF-1 was described previously.32,33

Nuclear/cytoplasmic isolation

Nuclear and cytoplasmic cellular fractions were isolated by utilizing the Pierce Thermo Scientific NE PER nuclear and cytoplasmic extraction kit (Pierce Thermo Scientific, Rockford, IL). Lamin A and GAPDH antibodies were utilized to evaluate the purity of the nuclear and cytosolic fractions respectively (Cell Signaling Technology Inc., Danvers, MA).

Luciferase assay

SK-BR-3 cells were plated in duplicate in 6-well plates and were co-transfected with NF-κB responsive luciferase reporter construct pELAM-luc70 and the β-gal plasmid pIEPlacZ. Cells were infected 24 h later with the indicated adenovirus at a MOI of 10. After 24 h, luciferase activity was measured by the Autolumat™ LB953 luminometer (EG&G Flow Technology Inc., Nashua, NH) and normalized to β-gal expression to obtain a relative luciferase activity value.

Statistical analyses

Data are presented as means ± standard error of the mean or standard deviation with representative experiments depicted in each figure. For apoptosis assays, comparisons between values were computed using analysis of variance (ANOVA). Unpaired t test was utilized to compare the mean values between cell cohorts, e.g., TNF-α and Ad-IRF-1 + TNF-α.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Our gratitude to Xiao Yan Gong and Shubing Liu for technical assistance.

Funding

Grant support was provided by NIH grant CA098403 and the Susan G. Komen for the Cure grant BCTR0708040 (J Yim) and the Elsa U Pardee Foundation (E Pizzoferrato).

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