IFNgamma restores breast cancer sensitivity to fulvestrant by regulating STAT1, IFN regulatory factor 1, NF-kappaB, BCL2 family members, and signaling to caspase-dependent apoptosis

Abstract

Antiestrogens are effective therapies for the management of many estrogen receptor-alpha (ER)-positive breast cancers. Nonetheless, both de novo and acquired resistance occur and remain major problems in the clinical setting. IFNgamma is an inflammatory cytokine that induces the expression and function of IFN regulatory factor 1 (IRF1), a tumor suppressor gene that can increase antiestrogen responsiveness. We show that IFNgamma, but not IFNalpha, IFNbeta, or fulvestrant (ICI; ICI 182,780; Faslodex), induces IRF1 expression in antiestrogen-resistant MCF7/LCC9 and LY2 cells. Moreover, IFNgamma restores the responsiveness of these cells to fulvestrant. Increased IRF1 activation suppresses NF-kappaB p65 (RELA) activity, inhibits the expression of prosurvival (BCL2, BCL-W), and induces the expression of proapoptotic members (BAK, mitochondrial BAX) of the BCL2 family. This molecular signaling is associated with the activation of signal transducer and activator of transcription 1 and leads to increased mitochondrial membrane permeability; activation of caspase-7 (CASP7), CASP8, and CASP9; and induction of apoptosis but not autophagy. Whereas antiestrogen-resistant cells are capable of inducing autophagy through IFN-mediated signaling, their ability to do so through antiestrogen-regulated signaling is lost. The abilities of IFNgamma to activate CASP8, induce apoptosis, and restore antiestrogen sensitivity are prevented by siRNA targeting IRF1, whereas transient overexpression of IRF1 mimics the effects of IFNgamma treatment. These observations support the exploration of clinical trials combining antiestrogens and compounds that can induce IRF1, such as IFNgamma, for the treatment of some ER-positive breast cancers.

Figure 1

IFNγ restores, and IRF1 siRNA transfection impairs, ICI sensitivity in breast cancer cells. (a) Antiestrogen-resistant MCF7/LCC9 cells were seeded in 96-well tissue culture dishes and treated with 0–1000 IU/ml IFNγ for 6 days, at which time cell proliferation was measured using the CCK-8 reagent. (b) MCF7/LCC9 cells were seeded in 96-well tissue culture dishes and treated with 100 or 1000 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ for 6 days before measuring cell proliferation as in (a). (c) Antiestrogen-resistant MCF7/LY2 cells were seeded in 96-well tissue culture dishes and treated with 0–1000 nmol/L ICI in the presence (open circles) or absence (closed circles) of 100 IU/ml IFNγ for 6 days before measuring cell proliferation as in (a). (d) and (e) Antiestrogen-sensitive MCF7/LCC1 (d) and MCF7 (e) cells were mock-transfected (closed circles), or transfected with either non-silencing control (CTRLsi, closed triangles) or IRF1-specific (IRF1si, open circles) oligonucleotides one day prior to seeding in 96-well tissue culture dishes. Cells were then treated with 0–1000 nmol/L ICI for 6 days before measuring cell proliferation as in (a). Each inset in c-e shows a representative Western blot for expression of IRF1 and the GAPDH or β–actin loading control. In all panels, data are presented as relative optical density (OD 450 nm) and represent the mean ± S.E. for a representative experiment; at least three independent experiments were performed. *p<0.05 vs. control/vehicle treatment, ^#p<0.05 vs. ICI treatment, and ^&p<0.05 vs. CTRLsi transfection.

Figure 2

Restoration of ICI sensitivity by IFNγ induces apoptosis, requires the induction of IRF1, and induces nuclear localization of IRF1. (a) MCF7/LCC9 cells were transfected with either non-silencing control (CTRLsi, white bars) or IRF1-specific (IRF1si, black bars) oligonucleotides one day prior to seeding in 96-well tissue culture dishes. Cells were then treated with ethanol (EtOH) vehicle, 100 or 1000 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ for 6 days before measuring cell proliferation. Data are presented as relative optical density (OD 450 nm) and represent the mean ± S.E. for a representative experiment; at least three independent experiments were performed. *p<0.05 vs. control/vehicle treatment. (b) MCF7/LCC9 cells were transfected with either non-silencing control (CTRLsi, white bars) or IRF1-specific (IRF1si, black bars) oligonucleotides one day prior to seeding in 6-well tissue culture dishes. Cells were then treated with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ for 3 days before detecting apoptosis by measuring mitochondrial membrane permeability (MMP). Data are presented as percent of total cells positive for green fluorescence (indicative of MMP) and represent the mean ± S.E. for three independent experiments. *p<0.05 vs. control/vehicle treatment. (c) MCF7/LCC9 cells were transfected with CTRLsi or IRF1si one day prior to seeding in 6-well tissue culture dishes and treating with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ for 48 h. Cells were lysed and IRF1 expression was detected by immunoblot. GAPDH serves as the loading control. (d) MCF7/LCC9 cells were seeded onto 18 mm² glass coverslips one day prior to treatment with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ for 48 h. The cells were then fixed, permeabilized, stained for IRF1 and DAPI, and visualized by confocal microscopy. Nuclear IRF1 expression (red staining) is observed in cells treated with IFNγ.

Figure 3

Combined treatment with IFNγ and ICI increases ISRE transcriptional activity, and reduces NFκB transcriptional activity, in an IRF1-dependent manner. (a) MCF7/LCC9 cells were seeded in 12-well tissue culture dishes one day prior to transfection with plasmids encoding ISRE-Luc or p65-Luc in combination with the control phRL-SV40-Renilla. Cells were then treated with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 500 IU/ml IFNγ for 48 h before lysis and analyis by dual-luciferase promoter-reporter assay. (b) MCF7/LCC9 cells were transfected with CTRLsi or IRF1si one day prior to seeding in 12-well tissue culture dishes, followed by transfection with ISRE-Luc and phRL-SV40-Renilla, treatment with vehicle or 500 IU/ml IFNγ for 48 h, and lysis/analysis as in (a). (c) MCF7/LCC9 cells were transfected with CTRLsi or IRF1si one day prior to seeding in 12-well tissue culture dishes, followed by transfection with ISRE-Luc and phRL-SV40-Renilla, treatment with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 500 IU/ml IFNγ for 48 hours, and lysis/analysis as in (a). (d) MCF7/LCC9 cells were seeded in 12-well tissue culture dishes one day prior to co-transfection with plasmids encoding p65-Luc, phRL-SV40-Renilla, and either pcDNA3-IRF1 wild type (WT) or the empty vector (EV) control. Cells were then treated with ethanol (EtOH) vehicle or 100 nmol/L ICI for 48 h prior to lysis/analysis as in (a). For a through d, data are presented as the ratio of luciferase-to-Renilla signal (Relative Light Units, RLU) and represent the mean ± S.E. for a representative experiment performed in quadruplicate. At least three independent experiments were performed. *p<0.05 vs. control, ^p<0.05 vs. IFNγ, and ^&p<0.05 vs. CTRLsi or empty vector (EV) transfection.

Figure 4

Combined treatment with IFNγ and ICI decreases the mRNA expression of pro-survival genes while increasing expression of pro-apoptotic genes in MCF7/LCC9 cells. (a) IRF1 mRNA expression is induced by IFNγ MCF7/LCC9 cells were seeded in T-25 cm² tissue culture flasks one day prior to treatment with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ for an additional 48 hours. Total RNA was extracted, reverse transcribed, and assayed for IRF1 expression by qRT-PCR. RPLP0 serves as the housekeeping gene. Data are presented as a ratio of IRF1:RPLP0 expression (relative mRNA levels) and represent the mean ± S.E. for a representative experiment; at least three independent experiments were performed. *p<0.05 vs. control/vehicle treatment. (b) BCL2, BCL-W, and survivin are reduced, while BAK and BAX are increased by IFNγ MCF7/LCC9 cells were seeded, treated, and assayed as in (a). Data are presented as a ratio of target gene:RPLP0 expression (relative mRNA levels) and represent the mean ± S.E. for a representative experiment; at least three independent experiments were performed. *p<0.05 vs. control/vehicle treatment.

Figure 5

Effects of IFNγ and ICI on the protein expression of pro-apoptotic and anti-apoptotic signaling molecules in MCF7/LCC9 cells. (a and b) Cells were seeded in T-75cm2 tissue culture flasks one day prior to treatment with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ for an additional 48 hours. Whole-cell lysates were prepared and analyzed by immunoblot for the indicated proteins; GAPDH serves as the loading control. Representative images are shown in (a), while densitometric analysis from at least three independent experiments is shown in (b). *p<0.05 vs. control/vehicle treatment. (c and d) IFNγ increases mitochondria-associated BAX. MCF7/LCC9 cells were seeded and treated as described in (a). Mitochondrial and cytosolic fractions were isolated and analyzed by immunoblot for BAX, GAPDH (loading control for cytosolic fraction), and cytochrome c oxidase IV (COX IV; loading control for mitochondrial fraction). A representative image is shown in (c), while densitometric analysis from at least three independent experiments is shown in (d). *p<0.05 vs. control/vehicle treatment, and ^p<0.05 vs. IFNγ.

Figure 6

Combined treatment with IFNγ and ICI inhibits the BCL2 P1 promoter and increases the activation of CASP7, CASP8, and CASP9 in MCF7/LCC9 cells. (a) MCF7/LCC9 cells were seeded in 12-well plates one day prior to transfection with different BCL2 promoter-reporter and phRL-SV40-Renilla constructs and treatment with 100 IU/ml IFNγ or 100 nmol/L ICI (singly or in combination), or ethanol vehicle for 48 h. Data are presented as Relative Light Units (RLU) and represent mean ± S.E. for a representative experiment; three independent experiments were performed. *p<0.05 vs. control, and ^p<0.05 vs. IFNγ. (b) Protein levels of CASP7, cleaved CASP7, CASP8, and cleaved CASP8 were detected by immunoblot in MCF7/LCC9 cell lysates after 48 h of treatment with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ A representative image is shown. (c) MCF7/LCC9 cells were seeded in white, 96-well tissue culture dishes one day prior to treatment with ethanol (EtOH) vehicle or 100 nmol/L ICI in the presence or absence of 100 IU/ml IFNγ Sixteen (CASP9) or 72 (CASP7, CASP8) hours later, caspase activity was detected using colorimetric or luminescent assay as described in the Methods. Data are normalized to the vehicle-treated control and represent the mean ± S.E. for three independent experiments. *p<0.05 vs. control, and ^p<0.05 vs. IFNγ.