BCL2 and CASP8 regulation by NF-kappaB differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells

Abstract

Resistance to endocrine therapies remains a major problem in the management of estrogen receptor-alpha (ER)-positive breast cancer. We show that inhibition of NF-kappaB (p65/RELA), either by overexpression of a mutant IkappaB (IkappaBSR) or a small-molecule inhibitor of NF-kappaB (parthenolide; IC(50)=500 nM in tamoxifen-resistant cells), synergistically restores sensitivity to 4-hydroxytamoxifen (4HT) in resistant MCF7/RR and MCF7/LCC9 cells and further sensitizes MCF-7 and MCF7/LCC1 control cells to 4HT. These effects are independent of changes in either cell cycle distribution or in the level of autophagy measured by inhibition of p62/SQSTM1 expression and cleavage of LC3. NF-kappaB inhibition restores the ability of 4HT to decrease BCL2 expression, increase mitochondrial membrane permeability, and induce a caspase-dependent apoptotic cell death in resistant cells. Each of these effects is reversed by a caspase 8 (CASP8)-specific inhibitor that blocks enzyme-substrate binding. Thus, increased activation of NF-kappaB can alter sensitivity to tamoxifen by modulating CASP8 activity, with consequent effects on BCL2 expression, mitochondrial function, and apoptosis. These data provide significant new insights into how molecular signaling affects antiestrogen responsiveness and strongly suggest that a combination of parthenolide and tamoxifen may offer a novel therapeutic approach to the management of some ER-positive breast cancers.

Figure 1.

Basal p65/RELA protein expression and the basal transcriptional activity of NFκB are both increased in antiestrogen-resistant MCF7/RR cells. A) Quantification and representative immunoblot of p65/RELA expression in MCF-7 and MCF7/RR cells. Whole-cell lysates (20 μg) were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted. β-Actin was loading control. Data are mean ± se relative p65:actin ratio (normalized to MCF-7 control cells) for 3 independent experiments. *P<0.05; Student’s t test. B) Basal transcriptional activity of NF-κB in MCF-7 and MCF7/RR cells. Data are mean ± se fold induction relative to MCF-7 cells for 4 determinations. *P < 0.001; Student’s t test.

Figure 2.

A) Par inhibits proliferation and synergistically restores 4HT sensitivity in antiestrogen-resistant MCF7/LCC9 and MCR7/RR cells. Cells were seeded in quadruplicate and treated with ethanol vehicle, 4HT (1 μM), or Par (500 nM) in the presence or absence of 4HT (1 μM) in CCS-IMEM for 7 d before counting. Data are mean ± se proliferation relative to ethanol-treated control of 4 determinations. P = 0.029 for MCF7/LCC9 vs. control; P = 0.015 for MCF7/RR vs. control. *P < 0.001, ^∧P = 0.004; ANOVA; RI = 3.3, 1.8, respectively. B–D) Par increases sensitivity to 4HT in antiestrogen-sensitive MCF7/LCC1 and MCF-7 cells (B, C) and synergistically enhances sensitivity to ICI 182,780 (fulvestrant) in TAM-resistant MCF7/RR cells (D). Cells were seeded in quadruplicate and treated with either 0–1000 nM (1 μM), 4HT (B, C), or 0–1000 nM ICI 182,780 (D) in the presence or absence of 500 nM Par in CCS-IMEM for 5 d before counting. Data are mean ± se proliferation relative to ethanol-treated control of 4 determinations. P ≤ 0.001 for all treatment groups; 1-way ANOVA. RI values for each treatment doseage are indicated. B) MCF7/LCC1 cells. *P ≤ 0.008; Student’s t test. C) MCF-7 cells. *P ≤ 0.001; Student’s t test. D) MCF7/RR cells. *P ≤ 0.029; Student’s t test.

Figure 3.

Par inhibits NF-κB-dependent transcriptional activity in all breast cancer cell lines. Cells were cotransfected with pNF-κB-Luc and pRL-SV40 Renilla constructs before treatment with 500 nM Par for 24 h before lysis and luminescence detection. Data are mean ± se relative luciferase:Renilla activity (relative light units) for 4 determinations. *P = 0.011, ^∧P = 0.016; Student’s t test.

Figure 4.

IκBSR (mutant IκBα; dominant-negative NF-κB inhibitor) expression sensitizes the resistant MCF7/LCC9 cells to antiestrogen 4HT and inhibits NF-κB dependent transcription. A) Characterization of MCF7/LCC9 cells stably expressing IκBSR and their EV controls. Whole-cell lysates (40 μg) from IκBSR stable transfectants and EV controls were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with mouse monoclonal FLAG-M5 and rabbit polyclonal IκBα antibodies, respectively. β-Actin was loading control. LCC9/IκBSR clone 5 cells overexpress IκBα/IκBSR and express FLAG, as shown in a representative immunoblot. B) IκBSR expression restores 4HT-induced inhibition of cell proliferation in MCF7/LCC9 cells. Cells were seeded in quadruplicate and treated with either ethanol vehicle or with 4HT (1 μM) for 7 d before counting. Data are mean ± se relative proliferation of 4 determinations. *P < 0.001; Student’s t test. C) IκBSR significantly inhibits NF-κB-dependent transcription in both MCF7/LCC9 and MCF7/LCC1 cells. Cells were transiently transfected in quadruplicate with pNF-κB-Luc and pRL-SV40 Renilla constructs with or without IκBSR for 24 h before lysis and luminescent detection. Data are mean ± se relative luciferase:Renilla activity (relative light units) for 4 determinations. *P = 0.001, ^∧P = 0.001; Student’s t test.

Figure 5.

Combined treatment with Par and 4HT induces apoptosis and enhances MMP in MCF7/LCC9 cells. Cells were treated with Par (500 nM), 4HT (1 μM), Par + 4HT, or ethanol vehicle in CCS-IMEM for 72 h before determination of apoptosis by annexin V assay (A) or for 18–20 h before measuring MMP (B). Data are means ± se normalized to ethanol-treated cells for ≥3 independent experiments. P = 0.042 (A), P=0.008 (B) for all treatment groups; 1-way ANOVA. *P < 0.05.

Figure 6.

BCL2 protein expression is down-regulated by the combined treatment with Par and 4HT in MCF7/LCC1 and MCF7/LCC9 cells. MCF7/LCC1 and MCF7/LCC9 cells were each treated with 500 nM Par, 1 μM 4HT, Par + 4HT, or ethanol vehicle in CCS-IMEM for 72 h before cell lysis. Whole-cell lysates (20–40 μg) were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted for BCL2. β-Actin was loading control. Data are means ± se from 3 independent experiments. A) Representative immunoblot for MCF7/LCC9 cells. B) Relative BCL2:β-actin ratio in MCF7/LCC9 cells. P ≤ 0.001 for all treatment groups, P = 0.032 for 4HT vs. control; 1-way ANOVA. *P = 0.003; ^∧P = 0.001. C) Representative immunoblot for MCF7/LCC1 cells. D) Relative BCL2:β-actin ratio in MCF7/LCC1 cells. P = 0.005 for all treatment groups; 1-way ANOVA. *P < 0.05; ^∧P < 0.05. −I, without caspase inhibitor (pancaspase or CASP8).

Figure 7.

Stable expression of IκBSR and siRNA to p65/RELA: both inhibit BCL2 protein expression in MCF7/LCC9 cells. A) Whole-cell lysates (40 μg) from LCC9/IκBSR cells (cells stably expressing IκBSR) and EV controls were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted for BCL2. β-Actin was loading control. Representative immunoblot is shown. Data are mean ± se relative BCL2:β-actin ratio normalized to EV controls from 3 independent experiments. *P = 0.029; Student’s t test. B) BCL2 protein expression is inhibited in resistant MCF7/LCC9 cells by siRNA p65/RELA NF-κB knockdown at 96 h. MCF7/LCC9 cells were seeded in 12-well dishes and were transfected with control (si-ctrl) or p65-specific (si-p65/RELA) oligonucleotides for either 24 or 96 h before lysis. Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted for p65/RELA and BCL2 as shown. GAPDH is loading control. Representative immunoblots for p65/RELA and BCL2 are shown.

Figure 8.

PI and the specific inhibitor to CASP8: both reverse the effects of NF-κB inhibition on cell proliferation and MMP in MCF7/LCC9 cells. Cells were seeded in quadruplicate and treated with 1 μM 4HT, 500 nM Par ± 4HT (1 μM), or ethanol vehicle in CCS-IMEM in the presence or absence of either a PI (A) or a CASP8 inhibitor (B) for 7 d before counting. Data are mean ± se relative proliferation of 4 determinations. A) PI fully reverses the effects of NF-κB inhibition on cell proliferation. P ≤ 0.001 for all treatment groups; 1-way ANOVA. *P = 0.029; ^∧P = 0.029. B) CASP8 inhibitor partially reverses the effects of NF-κB inhibition on cell proliferation. P ≤ 0.001 for all treatment groups; 1-way ANOVA. *P = 0.01; ^∧P ≤ 0.001. C) Cells were treated as above in the presence or absence of either a PI or a CASP8 inhibitor for 18–20 h before MMP was measured. Data are mean ± se relative MMP normalized to ethanol-treated cells for ≥3 independent experiments. *P = 0.012; ^∧P = 0.006.

Figure 9.

PI and the specific inhibitor of CASP8 both reduce sensitivity to the antiproliferative effects of 4HT and prevent mitochondrial membrane permeabilization in response to 4HT in LCC9/IκBSR cells (cells stably expressing IκBSR). A) LCC9/IκBSR cells were seeded in quadruplicate and treated with either ethanol vehicle, or with 4HT (1 μM) for 7 d in the absence or presence of either a PI or a CASP8 inhibitor before counting. Data are mean ± se relative proliferation of 4 determinations. *P = 0.014, 0.036; Student’s t test. B) LCC9/IκBSR cells were treated with either ethanol vehicle or 4HT (1 μM) for 18–20 h in the absence or presence of either a PI or a CASP8 inhibitor before MMP was measured. Data are mean ± se relative MMP normalized to ethanol-treated cells for ≥3 independent experiments. *P = 0.024; ^∧P = 0.037; Student’s t test.

Figure 10.

Combined treatment with Par and 4HT no longer inhibits BCL2 protein expression in the presence of PI in MCF7/LCC1 and MCF7/LCC9 cells. MCF7/LCC1 and MCF7/LCC9 cells were each treated with 500 nM Par or 1 μM 4HT, Par + 4HT, or ethanol vehicle in CCS-IMEM for 72 h in the presence of PI before cell lysis. Whole-cell lysates (20–40 μg) were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted for BCL2. β-Actin was loading control. Data are means ± se from 3 independent experiments. A) Representative immunoblot for MCF7/LCC9 cells. B) Relative BCL2:β-actin ratio in MCF7/LCC9 cells. P = 0.818 for all treatment groups; 1-way ANOVA. C) Representative immunoblot for MCF7/LCC1 cells. D) Relative BCL2:β-actin ratio in MCF7/LCC1 cells. P = 0.748 for all treatment groups; 1-way ANOVA.

Figure 11.

Combined treatment with Par and 4HT no longer inhibits BCL2 protein expression in the presence of a specific inhibitor to CASP8 (C8I) in MCF7/LCC1 and MCF7/LCC9 cells. MCF7/LCC1 and MCF7/LCC9 cells were each treated with 500 nM Par or 1 μM 4HT, Par + 4HT, or ethanol vehicle in CCS-IMEM for 72 h in the presence of C8I before cell lysis. Whole-cell lysates (20–40 μg) were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted for BCL2. β-Actin was loading control. Data are means ± se from 3 independent experiments. A) Representative immunoblot for MCF7/LCC9 cells. B) Relative BCL2:β-actin ratio in MCF7/LCC9 cells. P = 0.377 for all treatment groups; 1-way ANOVA. C) Representative immunoblot for MCF7/LCC1 cells. D) Relative BCL2:β-actin ratio in MCF7/LCC1 cells. P = 0.143 for all treatment groups; 1-way ANOVA.