Published OnlineFirst January 17, 2019; DOI: 10.1158/1541-7786.MCR-18-0481 Cell Fate Decisions Molecular Cancer Research Estrogen-Induced Apoptosis in Breast Cancers Is Phenocopied by Blocking Dephosphorylation of Eukaryotic Initiation Factor 2 Alpha (eIF2a) Protein Surojeet Sengupta1, Catherine M. Sevigny1, Poulomi Bhattacharya2, V. Craig Jordan2, and Robert Clarke1 Abstract Approximately 30% of aromatase-inhibitor–resistant, tion factor 4 and C/EBP homologous protein that facilitate estrogen receptor–positive patients with breast cancer ben- apoptosis. Notably, we recapitulated this phenotype of efit from treatment with estrogen. This enigmatic estrogen MCF7:5C in two other endocrine therapy–resistant cell lines action is not well understood and how it occurs remains (MCF7/LCC9 and T47D:A18/4-OHT) by increasing the elusive. Studies indicate that the unfolded protein response levels of phospho-eIF2a using salubrinal to pharmacolog- and apoptosis pathways play important roles in mediating ically inhibit the enzymes responsible for dephosphoryla- estrogen-triggered apoptosis. Using MCF7:5C cells, which tion of eIF2a, GADD34, and CReP. RNAi-mediated ablation mimic aromatase inhibitor resistance, and are hypersensi- of these genes induced apoptosis that used the same sig- tive to estrogen as evident by induction of apoptosis, we naling as salubrinal treatment. Moreover, combining define increased global protein translational load as the 4-hydroxy tamoxifen with salubrinal enhanced apoptotic trigger for estrogen-induced apoptosis. The protein kinase potency. RNA-like endoplasmic reticulum kinase pathway was acti- vated followed by increased phosphorylation of eukaryotic Implications: These results not only elucidate the mechanism initiation factor-2 alpha (eIF2a). These actions block global of estrogen-induced apoptosis but also identify a drugable protein translation but preferentially allow high expression target for potential therapeutic intervention that can mimic the of specific transcription factors, such as activating transcrip- beneficial effect of estrogen in some breast cancers. Introduction resistant breast cancer cells and xenografts (7–12). Using long- term estrogen-deprived (LTED) cells, such as MCF7:5C (7) and fi The bene cial effects of high-dose estrogen therapy in some MCF7/LTED (8), studies have confirmed that low doses of estro- breast cancers, the standard therapy for postmenopausal patients gen can induce intrinsic and extrinsic apoptotic activities (7, 8). with breast cancer prior to the introduction of tamoxifen, have MCF7:5C cells were derived from MCF7 cells by long-term culture – been widely overlooked until relatively recently (1 3). Recent in estrogen-free conditions (7, 13). Stress responses were activated clinical trials have reported a 30% to 50% clinical response rate during estrogen-induced apoptosis in MCF7:5C cells (14) and after estrogen therapy for heavily pretreated estrogen receptor contrasted with apoptosis induced by cytotoxic drugs such as – (ER) positive breast cancers that become resistant to aromatase paclitaxel (15). Therefore, it is important to determine the precise – fi inhibitors (AI; refs. 4 6). Comparable clinical bene t was also mechanism that induces apoptosis by estrogen in LTED breast – observed with lower doses of estrogen in AI resistant breast cancer. cancers (4). In MCF7:5C cells, studies suggest that ER and its classical in vitro in vivo Laboratory studies and have recapitulated the nuclear function is necessary to induce apoptosis (16). Planar beneficial effects of estrogen treatment in endocrine therapy– 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) 1Department of Oncology, Georgetown-Lombardi Comprehensive Cancer but induce apoptosis later because of a delay in the apoptotic 2 Center, Georgetown University Medical Center, Washington D.C. Breast trigger (18). Medical Oncology, The University of Texas MD Anderson Cancer Center, Unfolded protein response (UPR) and stress signaling play a Houston, Texas. critical role in endocrine-resistant breast cancers (19–22). Global Note: Supplementary data for this article are available at Molecular Cancer analysis of gene expression after E2 treatment in MCF7:5C cells Research Online (http://mcr.aacrjournals.org/). demonstrate that UPR, which follows an endoplasmic reticulum Corresponding Author: Surojeet Sengupta, Georgetown University, 3970 (EnR) stress, is involved in estrogen-induced apoptosis (14). UPR Reservoir Rd NW, Research Bldg. W 405B, Washington, DC 20057. Phone: 202- has three distinct adaptive pathways that are primarily cytopro- 687-7451; E-mail: [email protected] tective. These pathways are highly coordinated and act to mitigate doi: 10.1158/1541-7786.MCR-18-0481 the protein load using three sensors, namely inositol-requiring Ó2019 American Association for Cancer Research. protein 1 alpha (IRE1-a), activating transcription factor 6 (ATF6), 918 Mol Cancer Res; 17(4) April 2019 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst January 17, 2019; DOI: 10.1158/1541-7786.MCR-18-0481 UPR and Estrogen-Induced Apoptosis and protein kinase RNA-like endoplasmic reticulum kinase change in expression of transcripts was determined as described (PERK; ref. 23). However, failure to restore protein synthesis previously and used the ribosomal protein 36B4 mRNA as the homeostasis following prolonged EnR stress can lead to an internal control (33). Sequences of the primers used for ATF4, apoptotic cell death (24). Activation of the PERK pathway XBP1 (spliced), CHOP, PPP1R15A (GADD34), PPP1R15B increases phosphorylation of the serine-51 residue in eukaryotic (CReP), IRE1a and 36B4 genes can be found in Supplementary initiation factor 2 alpha (eIF2a) protein that inhibits global Table S1. protein synthesis and helps to attenuate the protein load within the EnR (23). However, sustained phospho-eIF2a–mediated Puromycin labelling translational repression can also initiate cell death (25). Dephos- One million MCF7:5C cells were plated in 10-cm diameter phorylation of eIF2a is catalyzed by two specific regulatory plates and treated with vehicle (0.1% ethanol) or 10 nmol/L subunits of protein phosphatase 1 (PP1) complex, known as 17b-estradiol for varying time. Puromycin labeling was per- GADD34 (growth arrest and DNA damage 34) and CReP (con- formed by adding 10 mg/mL puromycin for the last 10 minutes stitutive repressor of eIF2a phosphorylation; refs. 26–28). before harvest to assess global protein translation (34). Cells were Although GADD34 expression is induced by stress (29), CReP lysed using 1Â cell lysis buffer (Cell Signaling Technology) is constitutively expressed and maintains a balance in phospho- containing protease inhibitors (Roche Diagnostics) and phospha- eIF2a levels (28). We have investigated the role of downstream tase inhibitors I and II (EMD Chemicals Inc.). Whole-cell proteins targets of the PERK pathway in the context of E2-induced apo- were isolated and probed for total puromycinylated protein ptosis and examined whether this apoptosis response is replicated by Western blotting analysis. Western blot images were scanned by activating eIF2a signaling. and quantified using Image J software. The blot was stained with ponceau S prior to probing with antipuromycin to normalize Materials and Methods the puromycinylated proteins for each sample lane. Cell culture and reagents siRNA Cell culture media was purchased from Invitrogen Inc. and FCS Two specific ON-Target plus siRNAs were purchased for each was obtained from HyClone Laboratories. Breast cancer cells gene from Dharmacon Inc. targeting PPP1R15A (catalog no. MCF-7:5C (7, 30) and MCF7/LCC9 (31) were derived from MCF7 J004442-05 and J00442-08) and PPP1R15B (catalog no. cells. T47D:A18/4-OHT cells are derived from T47D:A18 cells, by J015013-05 and J015013-07). These siRNAs were used to long-term culture in presence of 4-hydroxy tamoxifen (32). Cells deplete protein levels of GADD34 and CReP protein were maintained in phenol red–free IMEM media supplemented levels, respectively. A nontargeting siRNA from Dharmacon with 10% charcoal dextran-treated FCS. The identities of all the Inc. (catalog no. D-001810-10) was used as a control. Trans- cell lines were authenticated using short tandem repeat profiling fection of siRNA was performed using Dharmafect I Reagent and cells were regularly tested for Mycoplasma contamination (Dharmacon Inc.), and the cells were harvested after 48 hours before and after completion of experiments. Salubrinal (catalog of transfection for protein extraction. no. 2347) was purchased from Tocris Bioscience. Puromycin was purchased from Thermo Fisher Scientific (A1113803). All the Western blotting analysis experiments were performed at least three times, in triplicate to Total proteins from whole cells were extracted using RIPA confirm the results. buffer containing protease inhibitors (Roche Diagnostics) and phosphatase inhibitors I and II (EMD Chemicals Inc.). Total Crystal violet cell density assay protein (15–25 mg) was run on the gels and transferred onto Cell growth assays were performed using a crystal violet assay. A nitrocellulose membranes. Membranes were subsequently total of 1.5 Â 104 cells were seeded in each well of a 24-well plate blocked with 5% nonfat dry milk in TBS and probed with primary and specific treatments