Approximately 30% of aromatase-inhibitor–resistant, estrogen receptor–positive patients with breast cancer benefit from treatment with estrogen. This enigmatic estrogen action is not well understood and how it occurs remains elusive. Studies indicate that the unfolded protein response and apoptosis pathways play important roles in mediating estrogen-triggered apoptosis. Using MCF7:5C cells, which mimic aromatase inhibitor resistance, and are hypersensitive to estrogen as evident by induction of apoptosis, we define increased global protein translational load as the trigger for estrogen-induced apoptosis. The protein kinase RNA-like endoplasmic reticulum kinase pathway was activated followed by increased phosphorylation of eukaryotic initiation factor-2 alpha (eIF2α). These actions block global protein translation but preferentially allow high expression of specific transcription factors, such as activating transcription factor 4 and C/EBP homologous protein that facilitate apoptosis. Notably, we recapitulated this phenotype of MCF7:5C in two other endocrine therapy–resistant cell lines (MCF7/LCC9 and T47D:A18/4-OHT) by increasing the levels of phospho-eIF2α using salubrinal to pharmacologically inhibit the enzymes responsible for dephosphorylation of eIF2α, GADD34, and CReP. RNAi-mediated ablation of these genes induced apoptosis that used the same signaling as salubrinal treatment. Moreover, combining 4-hydroxy tamoxifen with salubrinal enhanced apoptotic potency.

Implications:

These results not only elucidate the mechanism of estrogen-induced apoptosis but also identify a drugable target for potential therapeutic intervention that can mimic the beneficial effect of estrogen in some breast cancers.

The beneficial effects of high-dose estrogen therapy in some breast cancers, the standard therapy for postmenopausal patients with breast cancer prior to the introduction of tamoxifen, have been widely overlooked until relatively recently (1–3). Recent clinical trials have reported a 30% to 50% clinical response rate after estrogen therapy for heavily pretreated estrogen receptor (ER)–positive breast cancers that become resistant to aromatase inhibitors (AI; refs. 4–6). Comparable clinical benefit was also observed with lower doses of estrogen in AI–resistant breast cancers (4).

Laboratory studies in vitro and in vivo have recapitulated the beneficial effects of estrogen treatment in endocrine therapy–resistant breast cancer cells and xenografts (7–12). Using long-term estrogen-deprived (LTED) cells, such as MCF7:5C (7) and MCF7/LTED (8), studies have confirmed that low doses of estrogen can induce intrinsic and extrinsic apoptotic activities (7, 8). MCF7:5C cells were derived from MCF7 cells by long-term culture in estrogen-free conditions (7, 13). Stress responses were activated during estrogen-induced apoptosis in MCF7:5C cells (14) and contrasted with apoptosis induced by cytotoxic drugs such as paclitaxel (15). Therefore, it is important to determine the precise mechanism that induces apoptosis by estrogen in LTED breast cancer.

In MCF7:5C cells, studies suggest that ER and its classical nuclear function is necessary to induce apoptosis (16). Planar class I estrogens such as estradiol (E2) induce apoptosis within 48 hours of treatment (15). Class II estrogens like bisphenol initially act as an antagonist during the first week of treatment (17) but induce apoptosis later because of a delay in the apoptotic trigger (18).

Unfolded protein response (UPR) and stress signaling play a critical role in endocrine-resistant breast cancers (19–22). Global analysis of gene expression after E2 treatment in MCF7:5C cells demonstrate that UPR, which follows an endoplasmic reticulum (EnR) stress, is involved in estrogen-induced apoptosis (14). UPR has three distinct adaptive pathways that are primarily cytoprotective. These pathways are highly coordinated and act to mitigate the protein load using three sensors, namely inositol-requiring protein 1 alpha (IRE1-α), activating transcription factor 6 (ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK; ref. 23). However, failure to restore protein synthesis homeostasis following prolonged EnR stress can lead to an apoptotic cell death (24). Activation of the PERK pathway increases phosphorylation of the serine-51 residue in eukaryotic initiation factor 2 alpha (eIF2α) protein that inhibits global protein synthesis and helps to attenuate the protein load within the EnR (23). However, sustained phospho-eIF2α–mediated translational repression can also initiate cell death (25). Dephosphorylation of eIF2α is catalyzed by two specific regulatory subunits of protein phosphatase 1 (PP1) complex, known as GADD34 (growth arrest and DNA damage 34) and CReP (constitutive repressor of eIF2α phosphorylation; refs. 26–28). Although GADD34 expression is induced by stress (29), CReP is constitutively expressed and maintains a balance in phospho-eIF2α levels (28). We have investigated the role of downstream targets of the PERK pathway in the context of E2-induced apoptosis and examined whether this apoptosis response is replicated by activating eIF2α signaling.

Cell culture and reagents

Cell culture media was purchased from Invitrogen Inc. and FCS was obtained from HyClone Laboratories. Breast cancer cells MCF-7:5C (7, 30) and MCF7/LCC9 (31) were derived from MCF7 cells. T47D:A18/4-OHT cells are derived from T47D:A18 cells, by long-term culture in presence of 4-hydroxy tamoxifen (32). Cells were maintained in phenol red–free IMEM media supplemented with 10% charcoal dextran-treated FCS. The identities of all the cell lines were authenticated using short tandem repeat profiling and cells were regularly tested for Mycoplasma contamination before and after completion of experiments. Salubrinal (catalog no. 2347) was purchased from Tocris Bioscience. Puromycin was purchased from Thermo Fisher Scientific (A1113803). All the experiments were performed at least three times, in triplicate to confirm the results.

Crystal violet cell density assay

Cell growth assays were performed using a crystal violet assay. A total of 1.5 × 104 cells were seeded in each well of a 24-well plate and specific treatments were started after 24 hours; media was changed every 48 hours. Cell growth was assessed after 5 or 6 days of treatment by washing the cells once with 1X PBS and subsequently stained with crystal violet followed by washing with deionized water. Cells stained with crystal violet were permeabilized by citrate buffer. Absorbance was measured at 562 nm using a spectrophotometer.

RNA isolation and real time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen) and RNAeasy Kit (Qiagen) according to the manufacturer's instructions. RT-PCR was performed as described previously (33). Briefly, cDNA was generated from 1 μg of total RNA in a total volume of 20 μL using high-capacity cDNA Reverse Transcription Kits (Applied Biosystems). Subsequently, the cDNA was diluted to 500 μL and RT-PCR was performed using ABI QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems). Each well contained 10 μL of PowerUp SYBR Green PCR Master Mix (Applied Biosystems), 500 nmol/L each of forward and reverse primers, and 5 μL of diluted cDNA in 20 μL final volume. The change in expression of transcripts was determined as described previously and used the ribosomal protein 36B4 mRNA as the internal control (33). Sequences of the primers used for ATF4, XBP1 (spliced), CHOP, PPP1R15A (GADD34), PPP1R15B (CReP), IRE1α and 36B4 genes can be found in Supplementary Table S1.

Puromycin labelling

One million MCF7:5C cells were plated in 10-cm diameter plates and treated with vehicle (0.1% ethanol) or 10 nmol/L 17β-estradiol for varying time. Puromycin labeling was performed by adding 10 μg/mL puromycin for the last 10 minutes before harvest to assess global protein translation (34). Cells were lysed using 1× cell lysis buffer (Cell Signaling Technology) containing protease inhibitors (Roche Diagnostics) and phosphatase inhibitors I and II (EMD Chemicals Inc.). Whole-cell proteins were isolated and probed for total puromycinylated protein by Western blotting analysis. Western blot images were scanned and quantified using Image J software. The blot was stained with ponceau S prior to probing with antipuromycin to normalize the puromycinylated proteins for each sample lane.

siRNA

Two specific ON-Target plus siRNAs were purchased for each gene from Dharmacon Inc. targeting PPP1R15A (catalog no. J004442-05 and J00442-08) and PPP1R15B (catalog no. J015013-05 and J015013-07). These siRNAs were used to deplete protein levels of GADD34 and CReP protein levels, respectively. A nontargeting siRNA from Dharmacon Inc. (catalog no. D-001810-10) was used as a control. Transfection of siRNA was performed using Dharmafect I Reagent (Dharmacon Inc.), and the cells were harvested after 48 hours of transfection for protein extraction.

Western blotting analysis

Total proteins from whole cells were extracted using RIPA buffer containing protease inhibitors (Roche Diagnostics) and phosphatase inhibitors I and II (EMD Chemicals Inc.). Total protein (15–25 μg) was run on the gels and transferred onto nitrocellulose membranes. Membranes were subsequently blocked with 5% nonfat dry milk in TBS and probed with primary and secondary antibodies. Specific bands were visualized using chemiluminescence (Thermo Fisher Scientific). Details of the antibodies used are given in Supplementary Table S2.

Statistical analysis

Statistical significance was estimated using two-tailed, unpaired Student t test for pairwise comparison wherever relevant. For multiple comparisons, one-way ANOVA with two-tailed post hoc Dunnett test was performed. A P value of <0.05 was considered as statistically significant.

Estrogen induces global protein synthesis and activates the PERK pathway of UPR in MCF7:5C cells

We investigated the time-dependent effects of E2 on global protein synthesis in MCF7:5C cells. As evident by the incorporation of puromycin during protein synthesis, a significant increase (P < 0.01 at 24 hours vs. vehicle) in the global protein synthesis rate was observed until 48 hours of E2 treatment. However, global protein synthesis was suppressed by 40% versus vehicle at 72 hours (P = 0.01) and 96 hours (P < 0.01) post-E2 treatment (Fig. 1A). Phosphorylated PERK and phosphorylated eIF2α expression levels were elevated after E2 treatment. The increase in phospho-eIF2α expression at 48 hours preceded the attenuated rate of protein synthesis seen at 72 and 96 hours. ATF4 protein is preferentially translated following phosphorylation of eIF2α (35, 36) and was elevated at the early time points following initiation of E2 treatment (Fig. 1B). In contrast, a significant increase (P < 0.05) in ATF4 mRNA was detected only after 48–96 hours of E2 treatment (Fig. 1C). Conversely, proapoptotic CHOP mRNA was increased 12-fold (P < 0.01) after 48 hours of E2 treatment; expression remained elevated until 96 hours (P < 0.01; Fig. 1D). CHOP protein levels were substantially upregulated after 72 hours, corresponding with the cleavage of PARP, an indicator of apoptosis (Fig. 1B). We also measured the mRNA levels of other UPR sensors, and found that E2 treatment upregulated IRE1α (P < 0.01 at 48 and 72 hours) and the ratio of spliced XBP1/total XBP1 levels (P < 0.01 at 48 hours) in a time-dependent manner (Supplementary Fig. S1A and S1B). ATF6 transcripts were significantly elevated (P < 0.01) at 48 and 72 hours after E2 treatment but declined at 96 hours posttreatment (Supplementary Fig. S1C).

Figure 1.

E2-induced apoptosis of MCF7:5C cells is triggered by high protein translation and activation of PERK pathway. A, A representative Western blot of puromycin-labeled total proteins after indicated time of estrogen (17β-estradiol; E2, 10 nmol/L) treatment. The graph on the right panel shows the percent of puromycin-labelled proteins (average ± SD, n = 3) versus vehicle-treated cells. The intensity of each lane was normalized by the ponceau S stained lanes. B, Assessment of PARP cleavage, PERK activation, and its downstream effectors (phospho-eIF2α, ATF4, and CHOP) by Western blotting analysis. C and D, Transcripts levels of ATF4 (C), and CHOP (D) were assessed using RT-PCR after indicated time of estrogen (10 nmol/L) treatment (**, P < 0.01 versus vehicle treatment).

Figure 1.

E2-induced apoptosis of MCF7:5C cells is triggered by high protein translation and activation of PERK pathway. A, A representative Western blot of puromycin-labeled total proteins after indicated time of estrogen (17β-estradiol; E2, 10 nmol/L) treatment. The graph on the right panel shows the percent of puromycin-labelled proteins (average ± SD, n = 3) versus vehicle-treated cells. The intensity of each lane was normalized by the ponceau S stained lanes. B, Assessment of PARP cleavage, PERK activation, and its downstream effectors (phospho-eIF2α, ATF4, and CHOP) by Western blotting analysis. C and D, Transcripts levels of ATF4 (C), and CHOP (D) were assessed using RT-PCR after indicated time of estrogen (10 nmol/L) treatment (**, P < 0.01 versus vehicle treatment).

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17β-estradiol upregulates GADD34 mRNA but the protein level is unaltered

PERK-mediated phosphorylation of eIF2α occurs during cell stress and upregulates GADD34 expression (29, 37, 38). GADD34 protein functions as a negative feedback loop by dephosphorylating eIF2α and promoting recovery from UPR by derepressing protein translation (29, 39). GADD34 expression is induced transcriptionally by ATF4 (38) and by enhanced translation of GADD34 mRNA. The latter happens via an alternate 5′ upstream open reading frame (uORF) that is inhibited during unstressed conditions (37, 40). We measured the time-dependent regulation of GADD34 and CReP mRNA and protein levels following estrogen treatment. GADD34 mRNA expression was significantly upregulated after 48 hours of E2 treatment (P < 0.01) and continued to increase with treatment time (72 and 96 hours; P < 0.01). In marked contrast, GADD34 protein levels remained unaltered at all the time points evaluated (Fig. 2A and B). No change in the mRNA or protein levels of CReP was observed after estrogen treatment (Fig. 2A and B).

Figure 2.

Estrogen induces GADD34 mRNA but not protein in MCF7:5C cells. A, Levels of GADD34 (protein product ppp1R15a gene) and CReP (protein product ppp1R15b gene) mRNA were measured by RT-PCR after indicated time of E2 (17β-estradiol, 10 nmol/L) treatment (**, P < 0.01 vs. vehicle treatment). B, Levels of GADD34 and CReP protein were assessed by Western blotting analysis after indicated time of E2 (17β-estradiol, 10 nmol/L) treatment. β-Actin was measured to confirm equal protein loading.

Figure 2.

Estrogen induces GADD34 mRNA but not protein in MCF7:5C cells. A, Levels of GADD34 (protein product ppp1R15a gene) and CReP (protein product ppp1R15b gene) mRNA were measured by RT-PCR after indicated time of E2 (17β-estradiol, 10 nmol/L) treatment (**, P < 0.01 vs. vehicle treatment). B, Levels of GADD34 and CReP protein were assessed by Western blotting analysis after indicated time of E2 (17β-estradiol, 10 nmol/L) treatment. β-Actin was measured to confirm equal protein loading.

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Salubrinal induces apoptosis in MCF7:5C, LCC9, and T47D:A18/4-OHT cells

E2-induced apoptosis in MCF7:5C cells was preceded by an increased phosphorylation of eIF2α. GADD34 and CReP, the regulatory subunits of protein phosphatase complex, are responsible for eIF2α dephosphorylation. Therefore, we tested whether inhibition of GADD34 and CReP can increase phospho-eIF2α levels and induce apoptosis in the absence of estrogen. Salubrinal, a pharmacologic inhibitor of both GADD34 and CReP (41), produced a concentration-dependent decrease in cell number in MCF7:5C cells (Fig. 3A) over a 6-day period. In LCC9 (27) and T47D:A18/4-OHT cells (32), which are resistant to both 4-hydroxy tamoxifen and fulvestrant (Supplementary Fig. S2A and S2B) and are insensitive to E2, we also observed a concentration-dependent reduction in cell number with salubrinal treatment (Fig. 3B and C). Phospho-eIF2α levels were elevated after 48 hours of salubrinal treatment in all cell lines, as were ATF4 and CHOP expression and PARP cleavage (Fig. 3D–F). Salubrinal treatment increased Annexin V-positive and PI–positive cells in MCF7:5C (Supplementary Fig. S3A and S3B), LCC9 cells (Supplementary Fig. S3C and S3D), and T47D:A18/4-OHT (Supplementary Fig. S3E and S3F).

Figure 3.

Salubrinal, concentration-dependently, decreases cell number and induces apoptosis in MCF7:5C, LCC9, and T47D:A18/4-OHT cells by elevating phosho-eIF2α. Cell number was measured after a 6-day treatment with indicated concentrations of salubrinal in MCF7:5C (A), LCC9 (B), and T47D:A18/4-OHT cells (C). Crystal violet method was used to assess the cell numbers and the data are represented as percent cell number of vehicle treatment. Level of PARP cleavage, phospho-eIF2α, ATF4, and CHOP was assessed by Western blotting analysis in MCF7:5C (D), LCC9 (E), T47D:A18/4-OHT cells (F), after 48 hours of treatment with vehicle or indicated concentrations of salubrinal. β-Actin was used to confirm equal protein loading.

Figure 3.

Salubrinal, concentration-dependently, decreases cell number and induces apoptosis in MCF7:5C, LCC9, and T47D:A18/4-OHT cells by elevating phosho-eIF2α. Cell number was measured after a 6-day treatment with indicated concentrations of salubrinal in MCF7:5C (A), LCC9 (B), and T47D:A18/4-OHT cells (C). Crystal violet method was used to assess the cell numbers and the data are represented as percent cell number of vehicle treatment. Level of PARP cleavage, phospho-eIF2α, ATF4, and CHOP was assessed by Western blotting analysis in MCF7:5C (D), LCC9 (E), T47D:A18/4-OHT cells (F), after 48 hours of treatment with vehicle or indicated concentrations of salubrinal. β-Actin was used to confirm equal protein loading.

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E2 and salubrinal similarly regulate UPR-related genes

E2 treatment in MCF7:5C cells induces several UPR genes (14). We studied the induction of some key UPR genes after 24 and 48 hours of estrogen and salubrinal treatment. A similar pattern of gene regulation was seen with E2 and salubrinal treatment. The magnitude of induction by estrogen and salubrinal was similar for ATF4 (P < 0.05 at 48 hours of E2 and P < 0.01 at 48 hours of salubrinal), PPP1R15A (P < 0.05 at 48 hours of E2 and P < 0.005 at 48 hours of salubrinal), IRE1α (P < 0.05 at 48 hours of E2 and P < 0.005 at 48 hours of salubrinal), and spliced-XBP1 (P < 0.05 at both 48 hours of E2 and salubrinal; Fig. 4A–D). Induction of CHOP mRNA was markedly higher (Fig. 4E) after salubrinal treatment (P < 0.01 at 24 and 48 hours) compared with E2 (P < 0.05 at 48 hours) treatment. Levels of PPP1R15B, PERK, and total XBP1 did not change significantly after E2 or salubrinal treatment (Supplementary Fig. S4A, S4B, and S4C).

Figure 4.

Similar upregulation of UPR genes by estrogen and salubrinal in MCF7:5C cells. Regulation of UPR genes ATF4 (A), PPP1R15A (B), IRE1α (C), XBP1 (spliced; D), and CHOP (E) was measured using RT-PCR after 24 and 48 hours of treatment with vehicle (Veh), 10 nmol/L estrogen (E2), or 25 μmol/L salubrinal (*, P < 0.05; **, P < 0.01 versus respective vehicle treatment).

Figure 4.

Similar upregulation of UPR genes by estrogen and salubrinal in MCF7:5C cells. Regulation of UPR genes ATF4 (A), PPP1R15A (B), IRE1α (C), XBP1 (spliced; D), and CHOP (E) was measured using RT-PCR after 24 and 48 hours of treatment with vehicle (Veh), 10 nmol/L estrogen (E2), or 25 μmol/L salubrinal (*, P < 0.05; **, P < 0.01 versus respective vehicle treatment).

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E2 and salubrinal activate the mitochondrial apoptotic pathway

We further investigated how E2 and salubrinal induced apoptosis. Previous studies have reported that E2-induced apoptosis is mediated by the intrinsic mitochondrial pathway, since this effect is dependent on increased expression of the proapoptotic proteins Bim and Bax (7). We confirmed that both Bim and Bax protein levels were elevated 48–72 hours after estrogen treatment (Fig. 5A). We next determined whether salubrinal-induced apoptosis in MCF7:5C and LCC9 cells uses the same mechanism. Indeed, BIM and BAX protein levels were elevated in a concentration-dependent manner after 48 hours of salubrinal treatment (Fig. 5B). We also measured levels of the proapoptotic protein BAK and antiapoptotic BCL2 and BCLXL proteins. No change in expression was observed after E2 treatment in MCF7:5C cells or after salubrinal treatment in MCF7:5C and LCC9 cells (Fig. 5A and B).

Figure 5.

Similar regulation of intrinsic (mitochondrial) apoptotic markers by estrogen and salubrinal in MCF7:5C cells. Protein levels of proapoptotic markers (Bim, Bak, and Bax) and antiapoptotic markers (Bcl2 and Bclxl) were measured after indicated time of 10 nmol/L estrogen (E2) treatment in MCF7:5C cells (A), and after indicated concentration of salubrinal treatment for 48 hours in MCF7:5C and LCC9 cells (B).

Figure 5.

Similar regulation of intrinsic (mitochondrial) apoptotic markers by estrogen and salubrinal in MCF7:5C cells. Protein levels of proapoptotic markers (Bim, Bak, and Bax) and antiapoptotic markers (Bcl2 and Bclxl) were measured after indicated time of 10 nmol/L estrogen (E2) treatment in MCF7:5C cells (A), and after indicated concentration of salubrinal treatment for 48 hours in MCF7:5C and LCC9 cells (B).

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Depletion of individual regulatory subunits, GADD34 and CReP, induces apoptosis in MCF7:5C cells

The regulatory subunits GADD34 and CReP are responsible for dephosphorylation of eIF2α. To determine whether inhibition of both GADD34 and CReP is necessary for increased phospho-eIF2α levels and apoptosis, we used siRNAs to deplete their expression in MCF7:5C cells. Two different siRNA sequences were used for each GADD34 and CReP. Depletion of either GADD34 or CReP led to PARP cleavage in MCF7:5C cells. Notably, CReP levels increased when GADD34 was depleted. The increase in phospho-eIF2α, ATF4, and CHOP was greater in CReP-depleted cells than in GADD34-depleted cells (Fig. 6).

Figure 6.

Depletion of GADD34 and CReP induces apoptosis in MCF7:5C cells. Assessment of PARP cleavage, phospho-eIF2α, ATF4, and CHOP by Western blotting analysis after 24 hours of siRNA-mediated depletion of either GADD34 or CReP in MCF7:5C cells.

Figure 6.

Depletion of GADD34 and CReP induces apoptosis in MCF7:5C cells. Assessment of PARP cleavage, phospho-eIF2α, ATF4, and CHOP by Western blotting analysis after 24 hours of siRNA-mediated depletion of either GADD34 or CReP in MCF7:5C cells.

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Effect of salubrinal is potentiated by 4-hydroxy tamoxifen cotreatment

E2-induced apoptosis is completely blocked by 4OHT in MCF7:5C cells (7, 18). We tested whether 4OHT has any modulatory effect on salubrinal-induced apoptosis in MCF7:5C and LCC9 cells. Treatment with 1 μmol/L 4OHT, in combination with suboptimal concentrations of salubrinal (1.6 μmol/L and 3.1 μmol/L), significantly (P < 0.05) potentiated salubrinal-induced cell death in both MCF7:5C and LCC9 cells (Fig. 7A and B). This effect was also evident when 1 μmol/L fulvestrant was used in combination with suboptimal concentrations of salubrinal (Supplementary Fig. S5A and S5B). These drug combinations did not produce any noticeable differences in the levels of phospho-eIF2 alpha, ATF4, or cleaved PARP; a moderate increase in CHOP protein expression was observed in the combination treated cells compared with salubrinal alone (Fig. 7C).

Figure 7.

4-Hydroxy tamoxifen potentiates apoptotic effect of salubrinal in MCF7:5C and LCC9 cells. Cell growth was measured using the crystal violet method, after a 6-day period of treatment with 4-hydroxy tamoxifen (1 μmol/L) and salubrinal alone or in combination as indicated in MCF7:5C cells (A) and LCC9 cells (B). 17β-Estradiol (10 nmol/L) was used as a control (*, P < 0.05). C, Assessment of PARP cleavage, phospho-eIF2α, total eIF2α, ATF4, and CHOP by Western blotting analysis, in MCF7:5C and LCC9 cells after 48 hours of treatment with 4-hydroxy tamoxifen (1 μmol/L) and salubrinal (25 μmol/L) alone or in combination as indicated. β-Actin was used to confirm equal protein loading.

Figure 7.

4-Hydroxy tamoxifen potentiates apoptotic effect of salubrinal in MCF7:5C and LCC9 cells. Cell growth was measured using the crystal violet method, after a 6-day period of treatment with 4-hydroxy tamoxifen (1 μmol/L) and salubrinal alone or in combination as indicated in MCF7:5C cells (A) and LCC9 cells (B). 17β-Estradiol (10 nmol/L) was used as a control (*, P < 0.05). C, Assessment of PARP cleavage, phospho-eIF2α, total eIF2α, ATF4, and CHOP by Western blotting analysis, in MCF7:5C and LCC9 cells after 48 hours of treatment with 4-hydroxy tamoxifen (1 μmol/L) and salubrinal (25 μmol/L) alone or in combination as indicated. β-Actin was used to confirm equal protein loading.

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This study establishes activated PERK-mediated phosphorylation of eIF2α as a key regulator of estrogen-induced apoptosis in susceptible breast cancer cells. This apoptotic response is phenocopied by inhibiting dephosphorylation of eIF2α in breast cancer cells with acquired resistance to estrogen and antiestrogen (Fig. 8). The PERK pathway of UPR activates downstream signaling cascades that can restore proteostasis and may function as a prosurvival signal (23). However, prolonged and unmitigated activation of the PERK pathway can induce apoptosis (24, 42–44). A previous study, using MCF7:5C cells, confirmed that PERK-mediated phosphorylation of eIF2α is critical for estrogen-induced apoptosis as pharmacologic inhibition of PERK blocked estrogen-induced eIF2α phosphorylation and apoptosis (14). In addition, we reported that estrogen-induced apoptosis is a delayed process that distinctly contrasts with paclitaxel-induced apoptosis in the same cells (15). However, the underlying mechanism responsible for this temporal lag was unclear. Here, we show that the delayed trigger coincides with the time needed to accumulate proteins after E2 treatment. Thus, a threshold level may be required to initiate EnR stress and activate the UPR. In MCF7:5C cells, E2 stimulates global protein synthesis at early time points, but this is followed by a significant suppression of protein translation after approximately 48 hours. This temporal pattern of global protein synthesis coincided with high PERK phosphorylation and a subsequent increase in phosphorylated eIF2α, a direct target of PERK. Phospho-eIF2α can then attenuate global protein synthesis (45, 46). The timing of this trigger was consistent with our previous reports, where E2-induced apoptosis was rescued by 4OHT at early time points but not with ≥48 hours of estrogen treatment (15).

Figure 8.

Mechanism depicting estrogen-induced apoptosis in MCF7:5C cells and the apoptosis, which occurs with pharmacologic (salubrinal) and genetic inhibition (siRNA) of GADD34 and CReP that mimics estrogen action.

Figure 8.

Mechanism depicting estrogen-induced apoptosis in MCF7:5C cells and the apoptosis, which occurs with pharmacologic (salubrinal) and genetic inhibition (siRNA) of GADD34 and CReP that mimics estrogen action.

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Expression of GADD34 increases after eIF2α phosphorylation and acts as a feedback inhibitor of eIF2α dephosphorylation (29, 39). In MCF7:5C cells, despite a dramatic E2-induced increase in GADD34 mRNA, its protein levels remain unaltered (Fig. 2A and B). This observation could reflect a critical event that renders the cells unable to use feedback to reduce the phospho-eIF2α levels and restore proteostasis. Consequently, cells would experience a prolonged and unabated UPR, perhaps sufficient to induce apoptosis. Translation of GADD34 is preferentially enhanced under stress conditions (37), by ribosome reinitiation at the 5′ untranslated region that ensures high GADD34 protein levels (40, 47) and the recovery of repression of protein synthesis (47). In MCF7:5C cells, it is likely that this mechanism is compromised during E2 treatment resulting in loss of this feedback mechanism (Fig. 8). Further studies are warranted to understand this mechanism precisely.

E2-induced apoptosis is blocked by inhibition of the c-src activity in MCF7:5C cells (16). Multiple cellular functions are regulated by c-src (48) including estrogen-induced growth in MCF7 cells (49). Src inhibition leads to suppressed AKT phosphorylation in MCF7:5C cells (50). Because activated AKT influences E2-induced ER binding at the genome level (51), inhibition of c-src could regulate E2-induced transcription, reduce protein load, and fail to reach the threshold required to trigger sustained UPR and apoptosis in E2-treated MCF7:5C cells. Conversely, E2 and triphenylethylenes show a differential delay in triggering apoptosis (18). For example, bisphenol induces apoptosis in MCF7:5C cells on day 5 as compared with day 2 with E2 treatment (17, 18). We also showed that bisphenol is not as effective in recruiting either ER or its coactivators to the promoter of the estrogen-responsive gene TFF1. Moreover, the levels of TFF1 mRNA were markedly lower as compared with E2 treatment (17). Because of its diminished estrogenic property, bisphenol may take longer to accumulate the threshold levels of unfolded proteins required for PERK activation leading to a delayed trigger and apoptosis in MCF7:5C cells.

Elevated levels of phosphorylated-eIF2α preceded E2-induced apoptosis in MCF7:5C cells. Hence, we examined whether inhibiting the regulatory subunits of the PP1α enzyme complex (GADD34 and CReP) that facilitate dephosphorylation of eIF2α (26, 28; Fig. 8) can increase the phospho-eIF2α levels. We also determined whether this inhibition is sufficient to initiate a signaling cascade similar to E2 in MCF7:5C cells, and so induce apoptosis. We used both salubrinal, a pharmacological agent known to inhibit GADD34 and CReP (41), and two different siRNAs each against GADD34 and CReP. Inhibiting GADD34 and CReP by salubrinal in MCF7:5C cells markedly elevated the phospho-eIF2α levels and also induced apoptosis as evident by PARP cleavage and decreased cell number after 6 days (Fig. 3A and B). Remarkably, induction of apoptosis by salubrinal was not restricted to MCF7:5C cells. LCC9 cells (31), and T47D:A18/4-OHT (32) that are not susceptible to E2-induced apoptosis (Supplementary Fig. S6) but are E2-independent and cross-resistant to tamoxifen and fulvestrant, also undergo apoptosis after salubrinal treatment (Fig. 3). Comparing the individual role of GADD34 and CReP using siRNA-mediated knockdown revealed that depletion of either gene product induced apoptosis in MCF7:5C cells (Fig. 6). However, CReP depletion induced a more robust activation of phospho-eIF2α, whereas a partial compensatory mechanism was evident when GADD34 was depleted, as CReP protein was expressed at a higher level than the control siRNA-treated cells, and phospho-eIF2α was only marginally elevated.

The salubrinal-mediated increase in phospho-eIF2α was followed by elevated protein levels of ATF4, CHOP, and cleaved PARP in all three cell lines (Fig. 3D, E and F), implicating the same mechanism of apoptosis as occurs with inhibition of GADD34 and CReP, and with E2 treatment. We also found that salubrinal treatment did not significantly decrease the number of immortalized mammary epithelial cells (MCF10A cells; Supplementary Fig. S6) suggesting that it may not be toxic to normal mammary cells. Therefore, inhibitors of GADD34 and/or CReP that mimic E2-induced apoptosis may be potentially advantageous in the clinic. For example, these agents could extend the benefit of tumor regression to a wider patient population, including patients whose tumors are not inherently susceptible to E2.

The transcript levels of several signature stress-related genes that were previously reported to be regulated by estrogen in MCF7:5C cells (14, 16, 17) were similarly regulated by salubrinal alone in MCF7:5C cells (Fig. 4A–E). This observation indicates that these genes are regulated by the downstream effectors of phospho-eIF2α and may not be primary targets of estrogen. Indeed, most of the observed effects of E2 in MCF7:5C cells are likely driven by elevated levels of phospho-eIF2α.

The mechanism of estrogen-induced apoptosis in vivo was initially reported to be extrinsic (8) but was later shown to occur through the mitochondrion (intrinsic pathway; ref. 7) and followed later by the extrinsic mechanism (15). The role of mitochondrion-mediated apoptosis in estrogen-treated MCF7:5C was confirmed (7), as proapoptotic proteins BIM and BAX were upregulated but antiapoptotic proteins BCL2 and BCLXL were not altered. Notably, CHOP is a direct transcriptional inducer of BIM and can mediate apoptosis by EnR stress (42). Indeed, BIM protein levels increased after 48–72 hours of E2 treatment and preceded upregulation of CHOP (Fig. 5A). We found that in both MCF7:5C and LCC9 cells salubrinal treatment upregulated BIM and BAX protein whereas BCL2 and BCLXL remained unaltered (Fig. 5B), suggesting a similar mechanism was engaged as in E2-treated MCF7:5C cells. In LCC9 cells, pharmacologic inhibition of BCL2 induces apoptosis and alters the autophagy pathway in a way that prevents the ability to fuel the cellular metabolism using degraded products (52).

It is postulated that 5 years of E2-deprived environment is required in the clinic to reprogram breast cancer cells to induce apoptosis in response to subsequent E2 treatment (53). Targeting GADD34/CReP could be beneficial since sensitization to E2-therapy may not be required because GADD34/CReP inhibition also induces apoptosis in E2 nonsensitive ER-positive breast cancer cells. GADD34 knockout mice do not show any discernible differences in embryonic development or early adult life (27, 54); therefore, selective targeting of GADD34 may be less toxic than inhibiting both the regulatory subunits, GADD34 and CReP. Overall, this study defines the trigger of E2-induced apoptosis in susceptible breast cancer cells and provides a drugable target that can be potentially used for therapeutic intervention mimicking E2-induced apoptosis.

R. Clarke has ownership interest (including stocks and patents) in American Gene Technologies. No potential conflicts of interest were disclosed by the other authors.

The views and opinions of the author(s) do not reflect those of the US Army or the Department of Defense.

Conception and design: S. Sengupta, V.C. Jordan, R. Clarke

Development of methodology: S. Sengupta, V.C. Jordan

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Sengupta, C.M. Sevigny, P. Bhattacharya

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Sengupta, C.M. Sevigny, P. Bhattacharya, R. Clarke

Writing, review, and/or revision of the manuscript: S. Sengupta, C.M. Sevigny, V.C. Jordan, R. Clarke

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Sengupta

Study supervision: S. Sengupta, R. Clarke

Others (Provided cell lines to Dr. Sengupta to conform with the requirements of the referees): V.C. Jordan

The authors thank Karen Creswell and Dan Xun for their help at the Flow Cytometry Shared Resource at Georgetown-Lombardi Comprehensive Cancer Center. This work was supported by Public Health Service Awards (U54-CA149147 and U01-CA184902, to R. Clarke), Department of Defense Breast Program (W81XWH-18-1-0722, to R. Clarke), in part by GUMC Dean's Pilot Project Award (to S. Sengupta), and the Lombardi Comprehensive Cancer Center Support Grant (CCSG) NIH (P30 CA051008).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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